Team:METU Turkey/notebook.html

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Notebook

Click on the dates to read our lab notebook.

2012

December

2013

January

February

March

April

May

June

July

August

September

October

24.12.2012

30 plates are autoclaved. 2 of them is broken.

500mL agar prepared.

Oguz bought cotton, Buse ate harribo

Cleaned all the stuff.

Selin bought detergent and sponge for cleaning

We should use aliminium folio doubledeck

For labelling=iGEM 2013 and what is this? This must be written all of the stuff in the lab.!!

25.12.2012

Agars and broth put into +4 fridge.

28.12.2012

Plates moved to the fridge, strecthing.

Guys please when we are keaving the lab check all of the stuff windiws, bunsen burner gasses and all the things that belongs to our team.

04.01.2013

We organize our lab and put our equipments in our closet properly. All of the stuff are labelled.

04.03.13

Broth and agar autoclaved At 2:50 p.m

3 different competent cells are prepared in a broth.Mustafa hoca, Çağlar and C3.

Cells are in the shaker.

Plates are fulled with agar.

Thensterille stuffs our in the left closet the second shelf

05.03.2013

Cells are taken from the shaker.

Put 1 mL of cells 3 pieces 100 mL broth and put it into shaker.

Took stocks into eppendorfs.

06.03.2013

Arrived the lab. Open de closrt and some tips falled to floor, labelled and put in a box.

Our assistants enable us distilled water.

The dirty equipments are autoclaved.

Chemicals has arrived, labelled and put in the right places.

Emre, Özgün, Şeniz, Ilkem, Bilge, Begüm, Betül, Selin, Munta, Alişan, Hakan, Oğuz, Buse

- stocks have been prepared, chloramphenicol and kanamaycin are working.

-the plates that have been prepared,

C3, chl. amp. kan. Çağlar chl. amp. kan. Çağlar (new) chl. amp. kan.

Have been put into incubator at 8:50 p.m

-500 mL LB has been prepared, in the fridge.

-Buse have prepared LB and Agar.

- R.A Mustafa Çiçek's sample has been prepared again.

-measured the o.d of the samples. The results:

C3=0.301

Çağlar= 746

Competent cells are divided into two groups. The sample of Çağlar, is maden microcentrifuge at 4:30 p.m

At 5:20 CaCl2 is added, and let it cool.

Attention please! Do not leave Agars in -20 fridge. We lost all of the samples because of that mistake.

-I would kill youn if you could not find my eraser! ( Hakan )

-at 8:42 p.m the flasks are made ready for tomorrow morning to be autoclaved.

-the dirty equipments are leaved in the pot.

At 9:03 p.m plates are covered by parafilm and put into +4 fridge the place that iGEM METU Labelled,

Hood is cleaned by ethanol, sterilized by uv light and shutted down.

06.03.2013 Wednesday.

- C3 and Çağlar samples are taken from the shaker,

-25 microliter and 100 microliter cells spread into small and large plates.

-one of the glass spreader is broken

-The plates have been put into incubator at 5:20 p.m.

-the bunsen burner gases have been shutted down and checked, the bench cleaned with ethanol

07.03.2013

Chemicals are put into +4 fridge.calculations made for chemicals. Lab 206 enable us to get nuclease free water.

Antibiotics are prepared in these concentrations

Amp: 50mg/mL ( 1000X)

Kan: 50mg/mL (1000X)

Chl: 25 mg/mL (1000X) in ethanol

Tetr: 15mg/mL ( 1000X)

Note that chloramphenicol does not dissolve in water completely, that is why we are preparing the stock with 70% or more ethanol.

Also ampicillin does not dissolve in water completely, in the bottle it says dissolve in acid, Emre will search how we can prepare it.

We should be tidy by putting our stuffs in the closet. We had an accident today...

Tommorrow dh5alphas o.d is going to be measured. 2 from 05.03.2013

2 from 04.03.2013 and 2 fresh.

70% ethanol will be prepared

Dirty equipments are going to be autoclaved

08.03.2013

Measured the dh5alpha cells o.d . The value is 0.184

100 mL LB and 1 mL dh5alpha culture prepared in a flask.

Now we are trying to find a way to enter in the room that has shaker in it.

1:16 p.m the culture in the flask is put in the shaker, we borrow Eser Ünsaldı 's ID cart. It shoul be taken from the shaker after 2-4 hours.

At 3:43 p.m

80% glycerol has been prepared 50 mL in a falcon in +4 fridge.

The sample is take from the shaker. Spectrphotometer shows us Mustafa Çiçek's dh5alpha e.coli 0.013 ABS sonucunu verdi (wavelength 595 and 600nm). That makes us surprised and we take a deep breathe and hold it, prepare again.

18.03.2013

When we come to the lan, the bench were very dirty, we clean it with a detergent and ethanol.

Our team have a meeting with metumech (the student club for metu mechanical engineering student at metu). We are preparing over-night cultures.

19.03.2013

O.d results => 05.03 : 249

04.03: 1.351

Stock 150 dh5alpha 1954

Stock250 dh5alpha 0.003

Autoclave is done.

We shoul prepare the competent cells but the o.d values of dh5alpha cells are very low ( about 0.01). So we let the cells grow more in the shaker.

The second o.d value is 0.093 at 3:40 p.m

At 4 p.m AlperMutlu helps us to prepare antibiotics stock.

Amphicillin/ 100 mg /mL ( sodium salt, does not works with our water)

0.5gr/5mL prepared.

The one that we have dissolves in nh4oh

0.5 gr amp

5 mL distilled water in a nonstreile falcon

Mix the water and amp in the falcon

Vortex to dissolve completely

Filtred for strelizition

Kanamycin stock50Mg /mL

250mg/5mL prepared

Procedure is thensame as amphicillin

The cells taken from shaker the o.d value is 280

Tehy should be taken at 2 p.m. The measurements made in Gülay Ozcengiz's lab.

A beaker labelled as nonsterille because used as a waste

The values measured at 2 p.m Mustafa:195nm C3: 147nm Çağlar:340nm the measurements made in 595nm because we could not make it 600nm

We had been prepared cells three days ago and keep it at +4 fridge but when we do the measuremens again the o.d values are very low so, we learnt that we should not trust the fridge, the cells may be die because of the very low temperature.

20.03.13

Samet – 10:00

* “Çağlar” and “Other” labeled 2 broth were planted. (300ul)

* DH5alpha was planted in Amp+ plate (150ul)

* Broths are taken to shaker in 214 at 10:55.

* Agar is in the incubator in our lab at 10:48

Samet,Müge,Mehmet,Bilge,Selin

* 11:45 DH5alpha broth was prepared. (300ul/100ml)

* 500ml Lb was prepared.

* There is bacillus contamination in subculture

* 12:55 3 broths were taken from shaker in 214 and put in +4.

* Tetracycline stock/ 15mg/ml

75mg/5ml was prepared. Tetracyclin/distilled water. Procedure is same, it needs to be filtered.

* Chloramphenicol/ 25mg/ml

125mg/5ml was prepared

Chl/ %70 EtOH. Procedure is same it needs to be filtered.

* Filtration: near fire!

-sterile eppendorf (a lot)

-syringe (5)

-filter (0.45 um)

1) Sterile the eppendorfs using fire.

2) Put filter on syringe

3) Put syringe on the eppendorf and pour the antibiotic.

4) Press the syringe from behind. (not too much pressure! It should drip!)

5) Close, gelatinize and label.

-Do not fill the eppendorfs to the brim.

6) place the antibiotics in -20

Selin, Bilge, Irmak, Müge.

* We did gram (-) staining in the lab upstairs. (16:40) Waiting for air dry.

* Prepared ONC in the tubes.

* 2 x 5ml LB+(19.03.13 DH5alpha stock) single colony.

* 2x 5ml LB+(18.03.13 DH5alpha stock) 50ml ONC. (17:40)

Bilge

* Pink and purple lines of bacteria were observed in the stock. There are photos.

Şeniz+Irmak+Müge+Özgün+Emre+Oğuz+İlkem+Ekin

* We saw pink and purple bacteria in the gram staining. We think it is contamination.

* Colonies that were planted into LB from plates are going in 37.

17:00 we planted 150ul.

Selin,Oğuz,İlkem,Nilay,Müge,Irmak,Cansu

17:45 Özgün planted sub-cultures.

21:03.13

Dear iGEMers,

10:15 in the morning we took the ONCs with ilkem and analyzed them unter the microscope.

* We saw bacillus contamination.

* So we made autoclave. We autoclaved the pipettes in case. (the first pipette autoclave, I was so excited.)

* ?

* We did spring cleaning in the closets.

* The first ritually clean innocent will prepare alcohol!

13:00

* Mehmet+Bilge

* We took the stinky stuffs from autoclave.

P.S.:we might be out of paper towels.

- 500 ml EtOH was diluted.

P.S.: Autoclave is really smelly. Öğh.

25:03:13

17:30 Bolgi+Özgün+Begüm+Hakan+Şeniz+Ekin+Samet

And the others came for weekly meeting.

-We are preparing ONC.

- We are waiting for Selin for the shaker and keeping the cells at +4 meanwhile.

- DH5alpha’s are back in the -80

-We put ice in the freezer of +4

- we made a stabilization mechanism for the tube which will go into the shaker.

-benches are clean valves are closed. Loop is in the saucepan, stocks are in the -80 box.

- 18:30 tube is in the shaker.

- windows are closed we breathed.

17:55 we saw contamination in the stocks. We put single colony that we took from the plates into the shaker (18-18:10)

26.03.13

Emre, Müge, Mehmet

* 10:16 we took the ONC’s from shaker with TA Alper. There are junior biologists in the lab and I don’t know which lesson is this. – it turned out to be microbiology

* Sub-culture was prepared from ONCs (100ml LB/300 ul ONC labeled as A and B.)

* Planted flasks are taken to the shaker for 2 hours. (they will be out at 12:30)

* Fire and gas is closed, there is class but windows are closed anyways.

* 10:40 we left the lab.

11:00

Emre+Batu

* Flasks and LB’s are in the autoclave. (11:10)

* Bowls and jars are in the left closet on the 2nd shelf from the bottom.

* 11:19 we left the lab while taking the ONCs to gram control and we did the exit controls.

27.03.13

Oğuz

* I cleaned the bench and took the ONC’s from shaker.

* ! We have to find a solution for the cotton sticking the tubes!

* !There is a little bit more than it should be in the ONC B 2 labeled tube, pipette tip didn’t fit so I didn’t want to take the risk and put less.

* Clean autoclave machine’s cap doesn’t close properly, it caused trouble to other people so careful!

* I prepared the ONC’s as 300 ul/100 ml and put it into the shaker upstairs in the room with the spectro.

* Gases are closed, windows are closed, bench is clean. I breathed.

P.S. I had to pour the ONC B-2, the sticky cotton or whatever. A-2 is in the +4 (whatever is left)

- plates with 4 different antibiotics were prepared in the sterile hood.

17:00

Begüm, Özgün, Bolgi, Selin, Emre, Hakan,Batu,Samet,Müge,Mehmet,Burak,Şeniz,Ekin

* We came and took the clean autoclave.

* OD’s of A and B are good, around 0.700 (17:30)

* We started the competent cell protocol.

* We tried to use the new centrifuge in 107. It said Error(-1). This was around 19:00 and since the OD’s were increased we gave up from the competent cells.

* 19:15 new ONC was prepared, they will be out tomorrow.

* The bench is cleaned and the gases and the windows are closed.

Mehmet+Emre

* 14:00 LB agar 10gr/100ml was prepared.

* 14:10 LB agar is in the autoclave. Meanwhile the housekeeper took the trash out (writing in case of anything).

* Paper tower and napkin is needed!

* 14:40 sub-cultures are taken from shaker.

A sample: 0.214 B Sample: 0.233

* Samples were put back in the shaker (15:05) they will be out at 5.

* Clean autoclave is done.

* Plate was prepared. 100ul/100ml – Amp

25ul/100ml – Chl

15ul/100ml – Tet

50ul/100ml – Kana

* Eppendorf’s are autoclaved and 1:1 glycerol stock was prepared from the experiment tubes.

* When the samples’ OD reach 0.600 the competent cell protocol will be continued.

Emre

* 15:22 sterile hood was activated.

* 12:00 Emre+Batu, we died the ONC’s with gram and labeled as A and B. They are in thecloset on the non-sterile shelf.

* 12:51 Oğuz+Batuhan+Munta+Mehmet, we took the clean autoclave out and put them onto the sterile shelf. Sub-cultures are still in shaker.

* 12:57 Sub cultures are taken and transferred into tubs. Not to use big bottle of LB again and again we divided a 15ml falcon and labeled as “LB OD for blank” it is in the rack in the +4

* Be careful while closing the flasks with cotton.

* 13:15 Oğuz, Munta,Rıdvan and Mehmet went for OD. Şeniz and Begüm came.

* OD’s are; A sample: 0.222, B sample: 0.066, they will be put in the shaker again.

* Valves are good, windows OK, bench is clean, there is nothing on the outside (including the waste of the previous lab), we breathed.

15.19:

- OD for the sample A is 0.000 and B is 0.226.

- plasmids are taken and put into -20 nearby the antibiotics.

Lab is cleaned and checked.

The samples are put into the shaker once again to be taken at 17:30.

17.30

- the samples are taken from the incubator and OD is measured. A => 0.001 and B=> o.725

The sample B is divided into two. 100µL of the cells are spreaded onto plates and put into the incubator.

28.3.13

- the plates are taken from the incubator. There were colonies on the Chl+. No growth is observed in other plates. One of the plates without antibiotics showed a colony. Might be contamination.

- all plates are parafilmed and put into +4.

The lab is checked before leaving.

16.08

- the plates are checked and put back into the incubator for further growth.

- Stocks are put into -20.

- The pipette tips are not sterile anymore. Should be autoclaved.

- 70% ethanol should be prepared.

- LB is made.

- The lab is checked before leaving.

12:40

- The plates in the incubator is checked. Seems fine.

- The location of the rotor of the centrifuge is unknown.

- LB is prepared and autoclave is done.

17.00

- centrifuge is done at 4200 rpm for a longer period. (12 minutes)

19.24

- competent cell procedure is in process.

-100µLs of spread and streak plates are done on empty plates and plates with antibiotics.

21:50

Everyone has done at least one inoculation. The lab is cleaned and checked before leaving.

5.4.13

10.55

- the lab is occupied.

8.4.12

16.45

- the plates are forgotten in the incubator for a week. Nonetheless, thay are parafilmed and put into +4.

19.10

- human practice meeting is done. The lab is checked.

09.4.13

15.15

-250ml of LB agar is prepared.

- Autoclave is done.

-The lab is checked before leaving.

19:00

- 100mls of B.2.1 are inoculated onto plates with antibiotics . (5of them)

- One of them is contaminated.

19:50

- Put into the incubator.

- The lab is checked before leaving.

10.4.13

The plates are checked and they are put into the incubator again.

Autoclave is emptied.

- 15, 25, 50, 75 and 100 mg/ml of Chl plates are prepared.

To do list

- Autoclave should be done including the waste.

- Aluminium folio should be bought

- We need tissue paper

- Water, we need water

- We are not authorized to work on the bench in the back for this week.

- The lab is checked before leaving.

10.04.2013

- Autoclave is done.

-transformation is done. pGEM

-nuclease free water is dividen into two 15 mL falcons. Thay are at +4.

- 10 plates of Amp, 2 plates of Chl, 2 plates of Tet and 2 plates of Kan are prepared as well as empty plates.

- The competent cell protocol is revised.

- After pouring the plates, the hood is UV radiated and cleaned with alcohol.

- The pipette tips (100µL) are not sterile anymore. Thay are stored to be autoclaved again. A new box of tips is opened.

- The eppendorfs for the demonstration of the competent cell protocol is ready in the shelf of autoclaved things.

- We have extra distilled water in some bottles.

- Fresh ice is taken.

- Spreading procedure is completed at 10:45.

- The lab is checked before leaving.

19:10

- Put into shaker.

- 500 and 750µLs of DH5alpha stockB is inoculated (ONC)

- Plates with antibiotics are poured and labelled.

20:30

- ONCs are put into the shakers. They should be taken after 16 hours.

- The stocks that are accidentally removed from -80 are discarded into waste. ( competent cell C250 and Competent cell cağlar250)

- The eppendorf which was inoculated is discarded into waste.

- Streak plates are done witn ONC competents.

- 250 mL of LB agar is prepared.

o 150ml => 150µL Amp

o 25ml => empty

o 25ml => 6.25µL Chl

o 25ml => 3.75µL Tetr

o 25ml => 12.5 µL Kan

16:45

- The plates are put into incubator at 370C. They should be taken at 8am.

- The lab is checked before leaving.

19:15

- The dirty autoclave is done after the meeting.

- Research is being done

- Lab is cleaned.

- 11.04.2013

- The transformed bacteria are removed from the incubator. All of them meaning C3, ONC B2.2 and Çağlar are grown in the Amp plates. The plates are being parafilmed and put into +4.

15.4.13

- transformation protocol is revised.

250 ml LB agar is prepared.

Plates, 30 of them are UV radiated.

16.04.2013

10:30

- The ONCs are taken from the shaker.

- The competents are not resistant to antibiotics. (that's a good thing.)

- We observed the expected growth in Amp plates.

16:30

- The ONCs from the previous night is stocked with glycerin. They are at -80.

17.04.2013

Buse, Cansu, Hakan, Oğuz, Ekin

• Ekin said that, “ I took our transformation results then covered them with parafilm and put them into the fridge which is set to “+4” degree celcius to show the people.”

• Oğuz said that, “ We made the first stage of the transformation. We put the our ONC into the fridge which is set to “+4” degree celcius. We will control it later.

Bilge, Müge Mehmet

• We autoclaved laboratory equipments at 16:50.

• We take the equipments from the shaker and put them into the fridge which is set to “+4” degree Celsius.

• Distilled water is done.

• Last controls were made before leaving the laboratory, then we left the laboratory.

Bilge, Hakan, Oğuz, Cansu

• We started the transformation again and put LB into the shaker.

• We autoclaved laboratory equipments.

• We could not do positive control. All other controls were put into the incubator at 23:33.

• The transformation is done.

• All controls were made.

18.04.2013

Selin, Bilge, Duygu T.

• We took the bacteria from the incubator and checked them.

• We covered them with parafilm and stretch film.

• Put them into the fridge which is set to “+4” degree Celsius.

• Last controls were made before leaving the laboratory, then we left laboratory at 13:00

26.04.2013

• Last controls were made before leaving the laboratory, then we left the laboratory at 11:15

29.04.2013

• We did our regular meeting.

06.05.2013

• We put the guts, which were extracted by Babür, into the fridge which is set to “+4” degree Celsius at 16:40

• We took the plates from the fridge which is set to “-20” degree Celsius put them into the shaker.

• We took two 100 ml stocks from Babür’s examples, then we prepared four ONC. Two of them are 50 ml, other ones are 5 ml. Put them into the incubator.

• We put the other equipment into the cupboard.

• LB and Glycerol are in the frigde which is set to “+4” degree Celsius.

• Last controls were made before leaving the laboratory and we left the laboratory.

07.05.2013

• We stocked ONCs which were planted yesterday.

• We checked OD values

• OD values α1 0,355 α2 0,307 β1 0,395 β2 0,366

• We stocked these.

17.05.2013

• We put the plates into the shaker.(14:15)

• Last controls were made before leaving the laboratory and we left the laboratory.

23.05.2013

• Our kit-plates were came.

11.06.2013

• After the breakfast with the ITU IGEM Team members, we met them for laboratory training.

• We applied the transformation procedure and cleaned the bench over and over.

• Technically complex things told.

14.06.2013

• Cleanliness was made.

• Lab equipments were autoclaved.

• Locker was cleaned.

19.06.2013

• For the clean lab equipments, they were autoclaved.

• 23 agar plates were poured.

• We made the LB Broth (0,5 litres) and put them to the fridge (+4 degree celsius)

• Hood machine were not sterile in the light of that fact we could not make our protocol.

• Hood machine were Uved

• Last controls were made before leaving the labratorary.

24.06.2013

• 1 plate poured with tap water.

• Plates were autoclaved for the pouring.

• We poured the 19 plates with Chl.

• We made ONC with one stock and two component cells.

• We made cell culture to 5 agar plates, which contain CHL, with compenant cells strike

• All of the closing checks are OK

26.06.2013

• Transformation efficiency kit was tried.

• Equipments were autoclaved.

• Cells were put into the shaker.

• Plates were autoclaved.

• Cells were taken from shaker.

• Agar was poured to the plates.

• Transformated cells were cultured to the plates with Chl.

• The plates which are labelled “C4” put into the incubator.

• The agar levels of the plates were less than the normal therefore cell cultures could not be made

27.06.2013

• Autoclav machine is still broken.

• We used another laboratory to autoclave the equipments.

• We were thought that we could pour agar to 20 plates but due to the newly rosen problems we could only done 16.

• We prepared the cell cultures and parafilmed the rest of plasmids.

• Plasmids and the transformations were both put in the fridge (+4).

• Efficiency kits are in the fridge (-20) too.

• Plates are in the incubator.

09.09.2013

Batu, Şeniz, İlkem, Irmak, Özgün, Bolgi, Begüm, Emre

10:00

- ONCs are taken, transformations are in progress.

- K143012 (came from Başkent) will be transformed again.

- Supplies needed to make competent cell are given to Selin to autoclaving

11:30

- Ligation transformations are taken. Colonies were selected from K143012 1:1 and K143012 1:6 and planted as streak into Chl plates. They will be taken between 2-2:30 a.m.

- 3:1 is parafilmed and foiled in +4. ONC should be prepared.

- Ligation transformations are taken a few cells are found and planted as streak. They will be taken at 9.

- At 6, the latest ligation transformations will be taken.

- 1:1 and 1:6 streaks will be taken at 6, we are trying to see what will happen in the long run.

- ONCs of 3:1, dh5α-A, dh5α-B, ? , will be taken at 9 and competent cell and plasmid isolation protocols will be started.

- Exit controls are done (Emre, Batu, Özgün, Bolgi checking out!)

10.09.13

Irmak, Bolgi, Emre, Şeniz, İlkem, Müge, Samet, Begüm, Burak

09:00

- Competent cell is being prepared. It should be taken from shaker at 11:45

- Plasmid isolation of K302033+I732820 3:1 is done. Nanodrop value is 35.6. It is in -20 in eppendorf reck.

11:00

- K606040, 606013, 238027, 823003 digestions are made and put in incubator. They should be taken after 2 hours.

- We prepared alcohol, be careful while using.

- OD’s of competent are 0.090A and 0.059B. We put them back into shaker, they should be measured at 13:55.

11:25

- Clean autoclave is done.

13:55

- ODs are measured and 2 groups of competent cells are prepared and some of them (50ml) are labeled as topten1 dh5α-A1, B1 and C7. The other ones (35ml) are labeled as 2. 1’s are our priority.

- Ligation of K606013 and K 606040 are done as vector insert with the ratio of 3:1, 1:3. 1:1

- Speed thermocycle incubation is done for 1.05 min. K823003 and K 238027 transformations with the ratio of 3:1 and 1:1 will be taken at 13:00

- We are out of glycerol. We took from our advisor Ayça (?) we need to prepare a bottle of sterile glycerol ASAP.

- Ligation is done. 17:50 Left for overnight, will be taken at 09:50

- Sam8 is planted. K302033+I732820 3:1 transformation is done. (2ul and 5 ul) They will be taken at 16:30(?)

- K606040 and 606013 (1:1, 1:3 and 3:1) are transformed. They will be taken at 13:15

- C7 is planted for testing into 4 different antibiotics. They will be taken at 16:00(?)

- We couldn’t find empty plate so we couldn’t plant.

- K302033+I732820 1:1a, 1:1b, 1:6, 143053 digestions are made. We couldn’t see the band we were supposed to see in K302033+I732820 so it has only 302033, it didn’t ligate.

- We saw unrelated bands in 143053.

- We ran the morning digestions in gel, results are good.

- We prepared plates with Chl and put them into +4 room, we also moved the plates with Tet from Lab214 to +4 room.

- Sam8 is reliable. ONC should be prepared from 143053.

Instructions will be taken from Alişan!

11.09.13

Şeniz, Begüm, Samet, Bolgi

10:00

- Ligations are taken and deactivated according to the notes left by Alişan.

- Other parts (at -20) which are needed for ligation are checked to see if they are cut or not. Kanamycin backbone should be cut – is cut now.

- K823003 (S+P) + K238027 (X+P) (1:1 and 3:1) transformations are started.

- We prepared gel.

- K606013 transformation with C7 is started.

- Hakan came. Whole lab cheered up. He is so sexy and handsome. OH HOT!

Özgün, Burak, Hakan

- 4 ligations are taken at 15:45 and transformations are being done. They will be taken out at 20:00.

- Empty, Amp and Chl plates are prepared.

- 606013 competent testing 1:1 and 3:1, K823023+K238027 transformations are done. They should be taken out tomorrow morning between 8-9.

- Overnights of Sam8 and Başkent parts are prepared at 6. Sweeties are at the shaker on the right in the shaker room.

- At 19:00, 606040-606013, 1:1 and 3:1 transformations are taken out and planted as streak. They should be taken around 9-10.

- Bolgi’s extra transformations are taken, they are at +4.

- At 7:15, overnight of Emre’s Sam8 (again) is prepared (K238027), they will be taken around 9-10

- Trials of C7 are taken out, most of them are empty, some of them are contaminated and some of them have cellish things. We saw black cells.

*BOLGİ CAME*

- 4 ligation transformations are done they should be taken at 11.

- Bolgi broke the Başkent plate and made a new streak (the broken one was already contaminated with eggs and stuff in it) it will be taken at 11.

- 606040 and 606013 were supposed to be taken at 2:30 but we took them earlier and then put them back. They will be taken at 7. (This one is different than the ones done at 19.)

- Exit controls are done.

- Bangboss checking out!

12.09.13

Müge, Begüm, Irmak, İlkem, Batu, Bolgi, Emre, Şeniz.

Morning:

- Competent trials are taken out, C7 is good and working. It can be used.

- 11:30 Topten1 and 2 are planted on Amp, Chl and empty plates. They are in incubator, they will be taken out at 01:30

- 01:30 streaks will be taken.

- Plasmid isolations are done, measurements are: Sam8 K238027 – 30.1 and 49.7

Hakan:

- 2 falcons (K143053) are out in shatter at Lab 214 (at 5). They will be taken out tomorrow morning at 9.

- And I have done the exit controls.

Night:

- There is something black in Topten2 Amp/2. No, there is not something black in my eye and I do not make things up with my ass.

- There is growth in Amp1 and Chl1. We marked the area in which we observed growth at Chl2.

- We tried to select colonies from J04450 (Chl Backbone) streaks.

[To prevent the weird sesame plate we think that we shouldn’t shake the agar too much. The cause of sesame plate is still being investigated.]

- At 17:40 J04450 will be taken out of the incubator.

- At 19:30 ONCs of ligations will be taken.

Note for Selin: Alişan will come and check the PCR tomorrow.

P.S.: Transfer the ONC’s which should be taken at 19:30 to the shaker in the lab upstairs at 9:00 and check the heat just in case.

13.09.13

Irmak, Begüm, Müge

- 8 falcons from shaker room are transferred to the lab upstairs at 9:00. They should be taken at 19:30.

- Plasmid isolation of K143053 is done. Nanodrop values are: K143053-1:7.5 and K14053-2:3.6

- transformation of 143012 is done. It is in the shaker at Lab214. It should be taken at 13:30, will be centrifuged and planted.

- K143053’s plasmid isolation nanodrop values are too low so, ONC is prepared from streak plates. 16 hours will be up at 03:20.

- We talked with Mr. Necati, the guy in the chemistry has taken time off. We will go back at Monday around noon.

-Exit controls are done.

Kisses.

13.09.13

Hakan, İlkem

- Transformation of K143012 is put into the incubator at 13:55

- Controls are done.

Irmak, Begüm, Şeniz, Hakan, Burak (sick)

17:30

- We came to look at the plates and they are full of elliptic air balloons. We are scared. We went to ask Selin and came across our teacher Ayça. She said that ellipses are not cell colonies just air bubbles. She said that this could happen if we are using the agar plates before drying them properly. She said that we should have waited at least an hour after pouring agar into plates.

18:05

- We saw growth in only one of the streak plates of J04450.

- We prepared ONC with Chl LB (10ml/10ul). It should be out at 10:05 tomorrow morning. It is in back of the shaker at lab 214.

14.09.13

İlkem, Oğuz, Emre, Alişan, Şeniz, Begüm, Selin, Müge, Samet, Batuhan

- PCR is done at 10:25

Irmak, Begüm, Şeniz, Müge, Selin, İlkem, Bolgi

- PCRs are taken out at 12:45

- pcr, digestion, purification and ligation of K302033 +I732820 are done. Pcr was run as non purificated and purificated but the gel was connected reversely so no image was acquired.

- After the ligation (with kanamycin backbone) ofK302033 and I732820 they were transformed. 16 hours will be up at 15:15. (2ul and 5ul)

- AntimazF and Amp. Backbone was digested. AntimazF and Amp backbone (non purificated) were ligated together and 50ul AntimazF and Kanamycin backbone were ligated together.

Ratios: antimazF +Amp -1:1 and 1:4

AntimazF+Kan – 1:1 and 1:4

- Those were ligated and their transformations should be done.

- 143053 was planted as streak. They will be out at 14:30

- PCR, digestion and purification of K823003+K238027+Kan are done and they are transformed. They will be out at 22:30

- PCR of LacI is done again and it is run in gel.

15.09.13

Burak, Hakan, Ekin, Irmak

- 2 plates are taken from incubator at 14:30. Two ONCs from each one of them (K143053-Amp) and 2ONCs from K302033 (Chl) are made at 15:30. They are in the incubator on the right. They will be out Monday morning at 5.

- We have done the PCR’s. Nanodrop values are:

K302033 – 6.7 ng/ul

K606013 – 6.7 will be out at 16:30

K823003 – 3.3 will be out at 18:30

I732820 – 5.8

- Purification of PCRs are done. Non purificated and purificated states were run in gel at 19:50

Order of loading: PCR1-Pur1-PCR2-Pur2-PCR3-Pur3-PCR4-Pur4

- We autoclaved 1L agar and pipette tips at 21:00

- There were no bands at PCR1, we saw 3 bands at PCR2, single bands were observed at PCR3 and 4. There were no bands at purificated ones.

- Selin and Müge transformed K823023 into Bacillius and planted them into plate. (21.30-21:45). They will be taken around 18:30-18:45. After we planted Bacillius we saw sesame plates – before we planted the cells they were normal plates. We thought that we could distinguish sesames from colonies. (50,100,150 ul from 3 samples, 9 plates in total.)

We prepared antibiotic plates and put them into +4 room.

Kanamycin – 22

Tetracyclin – 6

Ampicilin – 18

- PCR product and PCR purif. were run in gel but the results are weird and also there is nothing in the PCR purif. so we are waiting for Alişan to come.

- We put the dirty spreader and flask of Bacillius into an aoutclave bag and put the bag in the waste cabinet.

- We will change the PCR program since the results are wrong. We are preparing the PCR’s which were done afternoon. Alişan asked us to change them like that:

1) 98 calcius degree 1min

2) 98 calcius 10sec

3) 60 celcius 30sec

4) 72 celcius 45sec

5) Go to 2 (?) 32

6) 72 celcius 10min

7) 4 celcius hold.

- We prepared overnight from plates of 823003 + 238027 at 03:00. They will be out at 18:00

16.09.13

Şeniz, Begüm, Emre, Batu, Özgün

- Plasmid isolations of K302033 and K143053 are done. They are at -20in grey reck.

- Nanodrop values are:

K302033 A:29.1

B: 31.4

K143053 :2.9

17.9.13

Morning:

Şeniz, burak, Begüm, Müge, Hakan, Irmak

- Bacillius transformations are taken at 08:00 and planted as 50,100,150 ul. They will be out around 23:30-00:30. (18 plates)

- At 9, plasmid isolation of Bolgi1 and Bolgi2 transformations are made.

- 11:00 gel is prepared at lab 214

- Nanodrop values of plasmid isolations are measured:

Antifazf-amp : 17,7

Antimazf – kan: 20.5

- 14:00 Özgün’s plates are taken out, they are at +4

- PCR purification of PCR products (4 tubes) from +4 are done. Nanodrop values:

Antimazf-15: 5.3

Antimazf- 27: 2.7

Sam8 – (?)

K606013(2) : 10.2 (peak at 230)

- Aside from Sam8; Mazf(E+S) (K302033) 10ul, 6ul dH2O

(X+P) K606013(1) 16ul

(E+S)K823003 12ul, 4ul dH2O

(X+P)I732820 16ul

(E+S)K606040 10ul, 6ul dH2O

(E+P) antimazf-15 15ul, 1ul dH2O

(E+P) antimazf-27 16ul

(X+P)K606013(2) 10ul, 6ul dH2O

2,5 cutsmart buffer, 0.75 enzim1, 0.75 enzim2. Completed to 20ul and digested. Other PCR products are at +4. They will be taken from incubator at 16:40

- Clean autoclave is done.

- To optimize the purification, we did PCR of antimazF+ kan. Backbone by completing to 50ul.

- 23:11 onc of sam8A and B are made. They will be out at 15:11

- In the morning Chl should be prepared with %70 ethanol (5mg/1ml ethanol). Then prepare plates with that Chl (250 ml agar) (?)

18.09.13

Bilge, Özgün

- Plates are prepared around 04:00, concentrations are 5 for Bacillius and 25 for E.Coli, labeled, in +4.

- Ligation transformations are planted (05:00), they will be out at 20:00

- Where are the spreaders? We could find only one, it would be very sweet if you could look.

- Clean autoclave was done.

- Where is the other yellow reck?

- No movement in Bacillus, we are keeping them in incubator.

-Around 18:00, 8 tubes of ONC (5Chl + 3Tet) were prepared. 19.09.13 at 10:00 they will be ready for plasmid isolation

- Chl backbone (linear) was cut (with E+P).

- From yesterday’s parts and today’s Chl parts;

K606040 + K606013+ Chl Backbone

K823003 + K606013 + Chl Backbone ligations are done. Transformation is prepared. 19.09.13 around 15:00 they will be ready for ONC preparation.

- Ligations of AntimazF Const. + Chl Backbone are done, transformations are prepared. 19.09.13 around 19:00 their ONCs will be prepared.

- We tried transformation with BC competent cells (B. Subtilis), K823025 and K823026 linearized backbones. 19.09.13 around 15:00 they will be checked.

- From the plates of;

K302033 + I732820 + Chl

AntimazF-15 + Chl

AntimazF-27 + Chl ONCs are prepared (12tubes). 19.09.13 around 16:00 they will be ready for plasmid isolation.

- Sam8 (K238027) is isolated, nanodrop values are;

Sam8 A = 65

Sam8 B = 73

- With the gel extracted parts and newly cut Chl linear vector ligated.

L1= PvegAntimazF+Chl Backbone

L2=K606040+K606013+Chl Backbone 19.09.13 at 13:00 they will be ready for transformation.

20.09.13

Morning:

- ONC’s are taken out of shaker at 12:15

- Planted plates are taken at 10:45 and their ONC’s were prepared.

- We prepared alcohol.

- Autoclave is done around 15:00.

- We prepared LB.

- 15:15 we prepared 7 PCRs and took them at 17:15.

Gel extraction was done, values are not promising.

- Ligations were put in at 16:30, they are taken at 18:40.

- Transformations of ligations will be done.

Night:

Irmak, Şeniz, Hakan; Alişan,Begüm

- 4 samples are taken from shaker at 23:50

- Ligations are done.

- Digestions will be out at 02:35

- K823003+Sam8+Ch Backbone and I732820+Chl Backbone are planted at 00:30. They should be taken at 16:30

- PCR was done, they will be out around 06:30-07:00

- Thermo should be taken at 07:00

D1: AntimazF + DT (29.6)

D2: Chl backbone (linear) (E+P)

D1+D2: AntimazF + DT + Chl Backbone

• Ligation was put in at 07:45. They will be out at 09:45 in the PCR in the nanodrop room.

** Ligation transformation was done. At 13:45 it will be continued with centrifuge and planting, in the shaker room next to ONC 2 tubes.

- PCR is taken it is running in gel.

- Polymerase from last year will not be used.

- Ligation will be taken out at 12:30 and transformed.

So far we have;

K606040 + K606013 + Chl 3 – 63,8

K606040 + K606013 + Chl 6 – 41,7

Phyps + AntimazF + Chl 11 – 72,1

Phyps + antimazF + Chl 16 – 93,1

Phyps + AntimazF + Chl 15 - 34,1

Phyps + AntimazF + Chl 14 - 202,7

21.09.13

Müge, Batu, Emre

- AntimazF + DT + Chl Backbone transformation is done. They will be taken out at 04:15 from incubator.

- We found 4 falcons of LB next to sink. Why were they there? We put them in +4.

- AntimazF + MazF const. transformation is done. It will be taken at 05:40 from the incubator in Lab107.

- I732820 + Chl transformation will be taken at 06:45 from the incubator.

- At 05:40 ONCs will be taken from the incubator across the door in the shaker room and their plasmid isolation will be done.

P.S.: All plates can be taken around 07:00-08:00 according to the colony situation.

- At 16:30 ONCs will be taken from the incubator which is in the middle of the bench in the shaker room.

- we prepared PCR from the parts we will send and run them in the gel and took pictures of them.

- We were out of eppendorf so we made clean autoclave.

- We started transformation of Bacillus but we couldn’t go through, there is nothing to do L

- Exit controls are done, good luck. Kisses.

22.09.13

Ekin, Begüm, Şeniz

- 5:30 ONCs are taken and their plasmid isolation was done. B0014 looked clear so we centrifuged and there were no cells therefore we couldn’t continue its isolation. K143012’s plasmid isolation was done.

K143012 (1) – 40,6

K143012 (2) – 15,3 they are in the isolated plasmid box.

- We checked the plates in the incubator, aside from the bubbles they were clean so we left them for growth. (around 7-8)

08:00 + Bilge (oha ilk defa adım geçiyo :D)

- AntimazF+DT+ Chl Backbone is full of colonies, yay!

- K143053, I732820+Chl Backbone 2ul,5ul, AntimazF+MazF (2ul,5ul) are back in incubator.

- AntimazF+Dt+Chl (2ul,5ul) was taken and its ONC was prepared. We parafilmed the plates and put them in +4. ONC was out in shaker room at 09:10 it will be out at 01:10.

- Exit controls are done.

25.09.13

Şeniz

- 9:50 nanodrop values of 6 eppendorf in +4 were measured.

1-2) 163 ng/ul -K143012

3) 305 ng/ul -K302033 + I 732820

5) 135 ng/ul - K606013

6) 161 ng/ul - K143053

7-8) 14 ng/ul - K238027

9) 145 ng/ul - K823003

- I did the digestions of those at 12:30, at 14:30 they will be set in 80 celcius for 20 min.

- I took Irmak’s ONC, it is in +4.

- 14:30 I put the digestions in 80 celcius.

- New digestions are put in at 15:30, at 17:30 they will be set in 80 celcius for 20 min.

- Ligations and gel extractions were done.

- Transformations were done.

- Ligations are in nanodrop room they will be taken in the morning.

1-2 3 5 6 7-8 9

1 ul sample + + + + + +

15 M.G.W + + + + + +

2.5ul cutsmart + + + + + +

0.75ul enzyme

27.09.13

Emre, batu, özgün,oğuz,samet,hakan

- ONC was prepared from Selin’s Bacillus (?) (cut,uncut) (Chl). It will be out at 15:00.

- ONC was prepared from stock bacillus they will be out at 14:00.

- Chl, Amp and Tet plates are prepared, we were out of Amp so we prepared that too.

P.S.: Gel tank and power supply are taken from Z-31, give them back!

- Bacillus were done streak as Selin wanted. Plates are in incubator(planted ones). Other plates are in the closet on the right. Planted ones will be taken from incubator at 17:00

- We assumed that Lig5 grew in a plate with tetracycline and prepared ONC from it. It is in the same beaker with bacillus ONCs. It is labeled as Lig5 ONC. It will be out at 17:00.

- Ligation 1,2,3,4,5 and 6 were transformed. They will be checked at 17:00. If there is colony growth ONC will be prepared, if there is not it will be checked in later hours and ONC will be prepared from it.

28.09.13

Irmak, Burak, Müge, Begüm, Ekin

- Ligation 1,2,3,4,5,6’s (2ul of each) planted plated will be out around 14:00-14:30 tomorrow. They are on the lower shelf in the 3tet, 3amp plates.(6 plates in total)

- Ligation 1,2,3,4,5,6’s (5ul each) planted plates will be taken tomorrow at 14:45. They are on the top shelf total 6 plates as 3tet and 3 amp.

Night:

Begüm, Ekin

- Lig5, bacillus cut, bacillus uncut and bacillus stock were taken from the shaker where they were forgotten. According to Alişan’s instructions Lig5, bacillus cut and uncut’s plasmid isolation are started.

- After 3 spins of centrifuge there were not much of a cell clamp but we assumed they were there and the isolation is being continued. Nanodrop values: lig5: -4, bacillus uncut(K823025): -7, bacillus cut (K823025): -5.5

- We took the forgotten plates out of the incubator. Bacillus are in material closet, İTÜ parts are in +4.

- Özgün’s ligation plates were taken at 00:30

- ONCs were prepared from the plates taken from incubator ,Özgün Lig3 5ul(Amp), Özgün lig4 5ul(Tet) and İTÜ J23104(Amp). They are in the shaker which is in the middle of the bench in the shaker room, 3 falcons will be taken at 16:00

- ONC was prepared from Bacillus stock plate at 02:40.It will be tken at 16:40 tomorrow. 1 falcon in the shaker on the left.

- -Exit controls are done.

- Kisses.

- Gel tank is still in the waste closet, we couldn’t take it since it was closed. Do not forget!

- Begüm and Ekin

18:30

- 0.2 mL of Bacillus stock ONC is inoculated into 10 mL of MNGE and put into the shaker.

19:50

- 4 ONCs of K302033 + I732820 + AntiMazF are put into the shaker in the middle. Thay should be taken at 11:50am.

- The ligations of Lig 2-5 and 7 are done.

The antibiotics are prepared such that you put one µL of any of them into one mL of agar. We do not know if it is the case for the antibiotics at -20.

21:53

- 11 plates are spreaded in total ant put into 370C.

29.09.13

- The cells in the incubator are not taken whereas it is 16:00 because no growth was observed.

16:00

- The three falcons ( 523102 Amp) are taken from the shaker.

- Tha Bacillus should be taken from the shaker at 16:40.

- Plasmid isolation is done:

1. 323102 ITU 10.3

2. Özgün Lig3 5µL Amp 10.5

3. Özgün Lig4 5µL Tetr 49.6

30.09.13

Morning

12:00

- The gel-tank is returned to Z-31.

- The two 15 mL and two 50ml falcons are taken from the incubator in the middle. Plasmid isolation is started.

- The ONC Bacillus K823025 uncut 100µL Chl 5+ might be contaminated. Be aware.

- The plasmid isolations of ONC 1, 2, 3, 4 K 302033 +I732820 +AntiMazF are done. They are put into +4 at 13:35. Nanodrop values can be measured at 2pm.