Team:Goettingen/NoteBook w11

Test PCR for specificity of DarRrev Primer/contamination of DarRrev PCR reactions

- Neg. control was not negative --> probably, primers or water are contaminated with M. smegmatis chrom.DNA (most likely because of order of pipetting the reactions) or they are non-specific generating another PCR product of the same size as DarRrev (seems very unlikely)

- Test PCR:

- 3 reactions, all reactions were pipetted individually (no mastermix)

- Old water from yesterday and old primer dilutions (1:20) from yesterday used (reaction A)

- Old water from yesterday and new primer dilutions (1:20) from today used (reaction B)

- New water from today and new primer dilutions 1:20 from today used (reaction C)

- All 3 reactions had the same composition as the neg. control reaction yesterday

- PCR program was the same as yesterday

- Gel run:

1%-agarose-1xTAE gel

Loading of 3 µl 2 log ladder as a marker

Loading of 4 µl PCR reaction (A, B, C or neg. control from yesterday’s PCR) + 1 µl 5xLD

Loading of 3 µl purified DarRrev PCR product (from yesterday) + 1 µl dH2O + 1 µl 5xLD

Run at 100 V

EtBr staining + destaining with water

UV detection

Loading: 2 log ladder/reaction A/reaction B/reaction C/neg. control from yesterday/purified DarRrev PCR product from yesterday/ 2 log ladder/ reaction A/reaction B/reaction C/neg. control from yesterday/purified DarR PCR product from yesterday/ 2 log ladder

-> The PCR product (ca. 630 bp + 2x 30bp = 690 bp) is only seen, when all old components are used, but not if new primers and old water are used --> old primer dilutions are contaminated --> dilutions thrown away

-> Since DarRrev-no template control was apparently positive because of not exchanging the pipet tip before adding the primers, the insert obtained from yesterday’s PCR will be further used for cloning…

Transformation of ligation reactions from 13.8.13 and re-trafo of part 6.2 C1 plasmid 

- E.coli XL1-Blue comp. cells used

- According to methods folder

- Neg. control contained 20 µl sterile dH2O

- For re-trafo of part 6.2 C1 plasmid, 1 µl of plasmid from purification on 9.8.13 was used

- For transformation of ligations, the whole ligation reactions were pipetted to the comp. cells

- After centrifugation, 500 µl supernatant were removed; strangely, the epi with pSB1C3+Prom1rev contained less than 500 µl solution (but cell pellet was at the bottom of the tube and the colour corresponded to that of LB + comp. cells seen in all other tubes… possibly less comp. cells?)

- Plating of all reactions on LBCm

- Incubation at 37 °C

Plasmid MiniPrep of part 8 C1

- Culture at > 4 ml…

- Elution

1st: 30 µl HPLC-water (pre-warmed) while incubating at 50 °C for 2 min

2nd: 22 µl HPLC-water (pre-warmed) while incubating at 50 °C for 2 min

Both elutions were collected in the same tube

- Concentration (NanoDrop)

Tidying up our – 20 °C freezer boxes…

- I found a tube where the date and the concentration where difficult to decipher. It was part 7 C1 plasmid. It could be the plasmid purified by myself on 12.7.13 with a concentration of 179.3 ng/µl. But since I wasn’t sure about that I measured the concentration (NanoDrop) again:

 - We still had the senseless RBS-GFP-Terminator clones; I threw them away, as well…

P3op + pSB1C3 C1: Cryo-Stock and MiniPrep

- preparation of DMSO cryo-stock (900 μl ON culture + 100 μl DMSO 100%, vortexing, put directly to – 80 °C in our box)

- Mini Plasmid Preparation of remaining ON culture using Nucleospin kit from Macherey-Nagel (I accidently used 750 μl A4 instead of 600 μl for washing of DNA on column); elution 1x with 30 μl HPLC-water (pre-warmed), incubation at 50 °C for 1 – 2 min)

- NanoDrop concentration measurement

 - stored in to-do-Box

DarRrev PCR – Gel run

- 1st try: loading of gel was difficult because of deformed wells --> some sample was spilled --> but this should be a semi-quantitative gel --> gel thrown away and new gel was poured and loaded

- 2nd try:

1% Agarose-1xTAE gel

loading of 3 μl 2 log ladder

loading of 4 μl of each PCR reaction + 1 μl 5xLD

run at 100 V

EtBr staining + destaining in water

UV detection

 Loading: Marker/Negative control/ 2 μl primer reaction/ 4 μl primer reaction/ 8 μl primer reaction/---

-> for 2 μl primer reaction and 4 μl primer reaction, a very weak band at ca 700 bp (approx. bp of DarRrev PCR product: 630 bp + 2x 30 bp = 690 bp) was detected

-> strength of this band seemed to decrease with increasing primer amounts

-> PCR did not work properly: M. smegmatis has high GC content, but normal HF buffer and PfuS were used --> try comercial Phusion® and GC buffer, only testing of 2 and 4 μl primer amount (lower concentration seem to be more optimal…

DarRrev PCR repeated – with Phusion® and GC buffer 

- Reactions pipetted as before (see 12.8.13, DarRrev PCR with normal PfuS and HF buffer)

- Volume 50 μl

- 4x Master Mix consiting of 5x GC buffer, water, Phusion® and dNTPs

- preparation of template (dH2O for neg. control, M. smegmatis chrom.DNA for other reactions), primers (iGEM_83, iGEM_84 and a part of water for 2 μl primer and neg. control reaction); then, addition of required volume of mastermix

- Testing of 2 and 4 μl primer; neg. control (dH2O as template and 2 μl as primers)

- PCR protocol: the same as for PfuS in HF buffer

1% Agarose-1xTAE gel

loading of 3 μl 2 log ladder

loading of 4 μl of each PCR reaction + 1 μl 5xLD

run at 100 V

EtBr staining + destaining in water

UV detection

Loading: Marker/Negative control/ 2 μl primer reaction/ 4 μl primer reaction

-> wells of gel were somehow damaged: this could explain weak band observed for neg. control (some of 2 μl sample ran over well into well of neg. control…) --> same gel as used for control of part6.2 C1 XbaI digest (see below and to check first DarRrev PCR using PfuS and HF buffer)

-> bands of expected product size for both reactions, 2 μl primers and 4 μl primers. But one cannot say, if 4 μl reaction contains really more product than 2 μl reactions since wells were so strange…

-> pool and purifiy 2 μl primer and 4 μl primer reaction, digest the PCR product and run it again with the neg. control on a proper gel

PCR clean-up of DarRrev 2 μl primers and 4 μl primer reactions 

- both reactions were pooled (ca. 100 μl)

- addition of 500 μl PB buffer

- elution with 1st 30 μl HPLC water (pre-warmed) while incubating at 50 °C for 2 min; 2nd 22 μl HPLC water (pre-warmed) while incubating at 50 °C for 2 min

- NanoDrop concentration

 - Stored in DarR reporter system-Box

EcoRI and PstI double digest of purified DarRrev PCR product 

- 30 μl DarRrev PCR product (purified today) + 3 μl EcoRI FD + 3 μl PstI FD + 4 μl 10x FD buffer = 40 μl digest reaction

- digest at 37 °C (heating block showed sometimes 37.7 or 37.8 °C though it was set to 37 °C) for 1 h

Gel run

- 1% Agarose-1xTAE gel

- loading of 3 μl 2 log ladder

- loading of 4 μl neg. control PCR reaction + 1 μl 5xLD

- loading of 3 μl of RD reaction + 1 μl 5xLD + 1 μl dH2O

- loading of 3 μl of uncut PCR product + 1 μl 5xLD + 1 μl dH2O

- (loading of samples for XbaI digest of part6.2 C1, that was already digested with EcoRI, as well – see below)

- run at 100 V

- EtBr staining + destaining in water

- UV detection

Loading: Marker/neg.control from PCR/uncut purified PCR product/ RD reaction of PCR product/ uncut plasmid part 6.2 C1/ RD reaction plasmid part 6.2 C1 E+ P digest, purified/---

-> Negative control is not negative (repeat neg. control PCR tomorrow, to see, if it’s really not negative, but purifiy and ligate DarRrev insert)

-> After R.D., PCR product is still seen (no over-digestion)

PCR clean-up of DarRrev PCR product

- With Qiagen PCR purification kit

- 500 µl PB buffer used

- Elution with 30 µl pre-warmed HPLC water, incubating sample for 2 min at 50 °C

- NanoDrop concentration measurement:

- Stored in DarR reporter system-Box

XbaI-digest of part6.2. C1 linearized with EcoRI 

- ca. 27 μl (mostly) linearized part6.2 C1 from 12.8.13

- addition of 4 μl FD buffer 10x, 4 μl XbaI FD, 5 μl dH2O

- in total: 40 μl

- digest for 2 h at 37 °C (after 1 h, it has been incomplete up till now…)

Gel run

- 1% Agarose-1xTAE gel

- loading of 3 μl 2 log ladder

- loading of 3 μl of RD reaction + 1 μl 5xLD + 1 μl dH2O

- loading of 1 μl of uncut plasmid + 1 μl 5xLD + 3 μl dH2O

- run at 100 V

- EtBr staining + destaining in water

- UV detection

Loading: Marker/uncut plasmid/ RD reaction

-> digest is still incomplete (linearized plasimd at ca. 3 kb), even after 2 h XbaI digest. Band of uncut supercoil plasmid very weak (runs at ca. 2 kb)--> purifiy vector for ligation

-> repeat gel run on proper gel (wells were apparently damaged; see DarRrev PCR gel run) --> same gel as used for control of DarRrev PCR reactions (see above; PCR using PfuS and HF buffer, but also the PCR reaction with Phusion® and GC buffer) --> apparently, the last part of the gel had damaged wells (fault of comb? Or because of multiple runs?)

PCR clean-up of XbaI- and EcoRI-digested part6.2 C1 plasmid 

- addition of 500 μl PB buffer

- elution with 1st 30 μl HPLC water (pre-warmed) while incubating at 50 °C for 2 min; 2nd 22 μl HPLC water (pre-warmed) while incubating at 50 °C for 2 min

- NanoDrop concentration

Repeat of gel run (see above)

- 1% Agarose-1xTAE gel

- loading of 3 μl 2 log ladder

- loading of 3 μl of RD reaction + 1 μl 5xLD + 1 μl dH2O

- loading of 1 μl of uncut plasmid + 1 μl 5xLD + 3 μl dH2O

- (loading of samples for DarRrev PCR, as well (see above))

- run at 100 V

- EtBr staining + destaining in water

- UV detection

Loading: Marker/neg.control from PCR/uncut purified PCR product/ RD reaction of PCR product/ uncut plasmid part 6.2 C1/ RD reaction plasmid part 6.2 C1 E+ P digest, purified/---

-> Same result as before (see above), but at least a bit nicer (though still smeary…) 

- Stored in DarR reporter system-Box

Hybridization of oligos for Promoter3rev, Promoter1rev and RBSrev 

- Oligos iGEM_73, iGEM_78 (RBSrev)

- Oligos iGEM_74; iGEM_79 (Promoter1rev)

- Oligos iGEM_76; iGEM_81 (Promoter3rev)

1. Oligos dissolved in required amount of HPLC water

2. 10 µl of each oligo of one hybridization pair were mixed

3. incubation for 10 min at 80 °C (for RBSrev oligos --> TM is below 80 °C) or at 98 °C (for Promoter oligos --> TM is above 80 °C)

4. after 10 min, heating blocks were switched off; the mixtures were left in the heating block until it was cooled down to ca. RT.

--> can be used directly for ligation

- Hybridizations stored in DarR reporter system-Box

- Oligos stored in Primer-Box

Ligation of

a) pSB1C3 E+P with DarR insert E + P

b) pSB1C3 E+S with Promoter1rev, Promoter3rev, or RBSrev

c) part6.2 C1 E+X vector with Promoter1rev or Promoter3rev 

Total volume of ligations: 20 µl

Ratio for DarRrev ligation: vector:insert = 1:3 --> for 50 ng vector of 2070 bp, use 50 ng insert of 690 bp (online ligation calculator)

Ligation a)

Ligation b)

Ligation c)

- All reactions were pipetted individually, no mastermix

- Incubation ON at 16 °C (cold room)

Transformation of ligation reaction from yesterday (pSB1C3 + Terminatorrev) 

- According to methods folder, but instead of removing 400 µl after plating 50 µl and centrifugation, 500 µl supernatant were removed

- Negative control: addition of 10 µl sterile dH2O

- Plating on LBCm  plates (35 µg/ml)

- Incubation at 37 °C

Preparation of LB Agar for Chloramphenicol plates

shopping and ordering in cellar

Trafo of Ribo A-D from yesterday´s ligation

protocol followed except for streak out

streak out in 3 steps: first 50 μl, spin down, than 500 μl to waste, resuspend and streak out again in 50 μl and the rest (ca. 150 μl ) on another plate

3 plates per transformation

6 transformation (4 Ribos (A-D), 1 religation, 1 negativ control with 20 μl H2O)

Gel and Nanodrop measurements of R.D. from friday (Jonathan)

Gel:

M|CFP|CFPr.d.|YFP|YFPr.d.|Part1|Part1r.d.|Part2|Part2r.d.|Part3|Part3r.d.|Part4|Part4r.d.|RiboA|RiboAr.d.

M|RiboD|RiboDr.d.|RiboC|RiboCr.d.

àRiboB was forgotten

Nanodrop:

New inoculation of part 8 in Lb Amp

Inocculation of GFP clones (part 6.2 clones 1-7)

for cryostocks and a masterplate, which was forgotten previously

Terminatorrev PCR – Gel ex

 -> Terminatorrev PCR from 7.8.13 looks a bit strange (band in neg. control at height of expected PCR product); it could be the primers, however, since the plasmid used as a template is still present in the sample, PCR clean up (as it was done) might not be enough to purifiy PCR product from template. Thus, gel extraction needs to be performed.

- Addition of <7.5 μl to each of the purified samples

- Loading of entire samples on 1 %-agarose-1xTAE-gel

- Loading of 2 log ladder (3 μl)

- Loading of part 7 C1 plasmid as a control (1 μl of 120.5 ng/μl + 1 µl 5xLD + 3 µl water)

- Run at 85 V

 - Gel extraction + purification using Qiagen PCR clean-up/gel extraction kit

- since Terminatorrev PCR product is ca. 190 bp, 1 volume isopropanol was added after the gel pieces were dissolved

- 2 columns used (loading of sample amounts corresponding to max. 400 mg gel in total per column); when loading one column, I pipetted some of the sample onto the TARA-tube by mistake… so this part of the sample was lost…

- elution from both columns with 30 μl HPLC water each into the same tube

- concentration (NanoDrop):

Terminatorrev PCR – Repeated

 - since PCR from 7.8.13 had a strange negative control, the PCR was repeated as on 7.8.13, but with some modifications:

- with only 2 μl primers (iGEM_71/72)

- 5 reactions in total:

a) neg. control 1: no primers --> which bands are caused by primers?

b) neg. control 2: no template --> which bands are caused by template?

c - e) 3 reactions with template and primers

- template: part 7 C1 plasmid, dilution from 7.8.13 used

- 6x MasterMix containing PfuS, 5x HF buffer, water and dNTPs

- after PCR: 3 reactions with template and primers pooled into 1 tube

- 1%-agarose-1xTAE gel (from Nina)

- loading of 3 μl 2 log ladder

- loading of 4 μl of each PCR reaction + 1 μl 5xLD

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

- samples ran almost out --> no conclusion possible --> gel run repeated:

- gel run:

- 1%-agarose-1xTAE gel

- loading of 3 μl 2 log ladder

- loading of 4 μl of each PCR reaction + 1 μl 5xLD

- (Loading of Terminatorrev PCR product digest, as well – see below; loading of digest of part 6.2 C1 plasmid with EcoRI, too – see below)

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> band observed in neg. control results indeed from primers --> one can work with the old PCR product

-> green PCR tube with pooled PCR reactions stored in 15 ml Falcon tube at – 20°C for purification

Restriction of Part 6.2 C1 plasmid with EcoRI to generate vector for ligation with hybridization oligos of promoters

1500 ng plasmid (6 μl of 261.5 ng/μl sample; purified on 9.8.13)

4 μl EcoRI FD

4 μl 10x FD buffer

26 μl water

in total 40 μl

 - digest for 1 h at 37 °C

- gel run:

- 1 %-agarose-1xTAE gel

- Loading of 3 μl 2 log ladder

- Loading of 1 μl plasmid (uncut) + 3 μl water + 1 μl 5x LD

- Loading of 3 μl RD reaction + 1 μl water + 1 μl 5x LD

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> digest is still incomplete

-> addition of 1 μl EcoRI FD, 1 μl 10x FD buffer, 8 μl water

-> digest for another 1 h at 37 °C

- gel run:

- 1%-agarose-1xTAE gel

- Loading of 3 μl 2 log ladder

- Loading of 1 μl uncut plasmid + 3 μl water + 1 μl 5x LD

- Loading of 4 μl RD reaction + 1 μl 5x LD

- (Loading of Terminatorrev PCR product digest, as well – see below; repeat of gel run of new Terminatorrev PCR, as well – see above)

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> Digest only slightly incomplete --> tolerable --> purify vector for further digest with XbaI with PCR clean-up kit

Purification:

 - Qiagen PCR purification kit

- 600 µl PB buffer used (by mistake…)

- Elution 1x with 30 µl HPLC-water (pre-warmed) with incubation at 50 °C for 2 min

- NanoDrop: concentration

- Samples stored in To-do-Box

Restriction of Terminatorrev PCR product obtained after gel ex with EcoRI and PstI (for ligation with pSB1C3)

30 μl PCR product

3 μl EcoRI FD

3 μl PstI FD

4 μl 10x FD buffer

40 μl in total

- digest for 1 h at 37 °C

- gel run:

- 1%-agarose-1xTAE gel

- Loading of 3 μl 2 log ladder

- Loading of 4 μl uncut PCR product + 1 μl 5x LD

- Loading of 4 μl RD reaction + 1 μl 5x LD

- (Loading of part6.2 C1 plasmid EcoRI 2 h digest, as well – see above; repeat of gel run of new Terminatorrev PCR, as well – see above)

- Run at 100 V

- EtBr staining + destaining in water

- UV detection

-> PCR product still present/ no over-digest --> PCR clean-up to purify insert for ligation

Purification: 

- Qiagen PCR purification kit

- 500 µl PB buffer used

- Elution 1x with 30 µl HPLC-water (pre-warmed) with incubation at 50 °C for 2 min

- NanoDrop: concentration

- Samples stored in To-do-Box

DarRrev PCR: Test-PCR for optimal primer concentration 

- DarRrev primers (iGEM_83 and iGEM_84) arrived --> dissolved in HPLC-water and preparation of 1:20 dilution in HPLC water

- iGEM_83 and iGEM_84 tend to form strong secondary structures --> test for optimal primer concentration in PCR

- Test- PCR done as on 24.6.13: 5x MasterMix containing water, 5x HF buffer, PfuS, dNTPs; 2 μl, 4 μl and 8 μl primer tested, neg. control w/o template (M.smegmatis chrom.DNA); Annealing temperature of both primers is the same as for iGEM_33 and iGEM_44, since only the prefix and suffix sequences were switched)

- PCR taken out and stored at 4 °C (small fridge) in yellow-tip-box-rag

Sequencing results of Promoter3op cloning in front of part 6 and into shipping vector 

-> Plasmids part 6.2. C1 and part 6.2. C2 contain the Promoter3op insert at the desired site in the vector w/o any mutations --> part 6.2 C1 used for further cloning

->Plasmid Promoter3op in pSB1C3 C1 (see: Colony PCR from 8.8.13) contains the Promoter3op insert at the desired site in the vector w/o any mutations --> inoculation of C1 in 4 ml LBCm for MiniPrep and CryoStock, incubation ON at 37 °C, ca. 200 – 210 rpm

Ligation of pSB1C3 (E+P-digested) with Terminatorrev insert (E+P digested)

- Ligation calculator: for ratio vector: insert= 1:3 and 50 ng vector (2070 bp), ca. 14 ng insert (190 bp) are required 

-> Ligation reactions incubated ON at 16 °C (cold room)

Preparation of LBCm plates 

- From 500 ml medium

- 35 µg/ml Cm in plates

Ligation of Riboswitch Constructs A-D into pSB1C3(from part 7)

For that, ~45ng Vector were ligated to ~150ng Inserts as follows:

                  1.5µl pSB1C3

                  4.5µl Insert (Ribo A-D)

                  2µl T4 Buffer

                  2µl T4 Ligase

                  10µl H20

         = 20µl reaction

Incubation overnight at 16°C