Team:UCL/Labbook
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Revision as of 17:15, 1 October 2013
June
Week 1-3
No lab work
Week 4
26th June - We are given safety training in the Advanced Centre for Biochemical Engineering in all relevant laboratories, as well as general procedures in case of emergency.
July
Week 5-6
No lab work
Week 7
Bacterial Lab
15th July - The team is introduced to the laboratories which will be used during the summer for both bacterial and mammalian experiments.
16th July - 5X M9 salts, minimal agar, 1.4% molten agar solution and 0.1M CaCl2/15% glycerol were prepared for the generation of competent cells. Minimal agar plates were poured and streaked with W3110 Escherichia coli cells and left overnight to incubate at 37C.
17th July - Very little colony growth was observed from W3110 ''E.coli'' streaked plates. Plates were therefore left to incubate for a further 17 hours.
18th July - Sufficient colony growth allowed for the selection of a single colony from each plate. This was then inoculated in 5ul LB media + 100ul 1M MgSO4 and left to incu-shake overnight at 37C.
19th July - Cultures re-suspended in new LB media and 100µl aliquots placed into individual eppendorf tubes for placement at -80C.
Mammalian Lab
17th July - Mammalian cell culture and maintenance training by Mrs. Ludmilla Ruban. Passaged primary MEF (mouse embryonic fibroblast) cells. Passage 3.
18th July - MEF passage 4
19th July - MEF passage 5
Week 8
Bacterial Lab
22nd July - Transformation of our competent cells with plasmid YB3110 was carried out.
23rd July - No colony growth was observed on Ampicillin plates indicating no plasmid uptake. transformation was repeated with YB3110.
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 0 |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 0 |
24th July - No colony growth from main experiment indicating no plasmid uptake. Transformation was repeated.
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 0 |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 0 |
25th July - No colony growth and hence no plasmid uptake. Transformation was repeated.
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 0 |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 0 |
26th July - Once again our cells were unsuccessful in taking up the YB3110 plasmid. We concluded that our batch of competent cells were not competent and so these will not be used in any further experiments. A new batch of competent cells are to be generated.
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 0 |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 0 |
22nd July - MEF passage 6. Training ends.
Week 9
Bacterial Lab
29th July - A glycerol stock of pSecTag2A from 2009 was restored. This was streaked onto 3 plates (2x LB Amp and 1x No drug), additionally four Falcons with 2ul LB were inoculated with the glycerol stock (2x LB No drug, 2X LB Amp). These were left to incubate overnight.
30th July - pSecTag2A Amp and No drug cultures were centrifuged (5 minutes at 4000rpm) and the pellet frozen to be used later in a miniprep. pSecTag2A plates displayed good colony growth. Colonies were picked from non-competent W3110 streaked plates and inoculated in LB ->incu-shake 37C o/n.
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 100+ |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 0 |
31st July - A new stock of competent cells were generated and stored, these were tested for competence via transformation using pSecTag2A and streaking onto amp plates -> incubate 37C o/n.
Ampicillin was produced and stored. Following miniprep of pSecTag2A a gel was prepared for analytical digest with HindIII [link to HindIII conditions] [insert gel image HindIII analytical digest of pSecTag2A]. 50X TAE diluted to 1X.
Item | Volume (ul) |
---|---|
DNA pSecTag2A | 5 |
HindIII | 1 |
Buffer | 1 |
BSA | 0.5 |
dH20 | 2.5 |
Total | 10 |
Results displayed uncut bands at expected lengths. However HindIII cut pSecTag2A wells displayed too many bands, indicating possible contamination or uncut DNA.
August
1st August - Results from newly generated competent cells transformed with pSecTag2A:
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 100+ |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 10 |
This indicated that the cells are more competent than the last batch, although growth on the negative control was a cause for concern. Therefore the transformation protocol from yesterday was repeated to see whether the ampicilin was working -> plates left for incubation 37C o/n.
Transformation of competent cells with pSecTag2A. 100µl was then spread onto 6 plates (4x Amp, 2x No drug). Left to incubate o/n 37C.
2nd August - Plates displayed significant colony growth on amp plates indicating successful transformation. Negative control however displayed slight colony growth - ampicillin not working effectively, new ampicillin was therefore prepared. End of week inventory was recorded and stocks were topped up.
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 100+ |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 15 |
29th July - Thawed and revived HeLa cells, grown in two T25 flasks in DMEM + 10% FBS + 2 mM L-Glu
30th July - HeLa cells are not looking very happy, with many floating cells. Cells are left to grow over the weekend
Week 10
Bacterial Lab
5th August - The new batch of ampicillin was tested via transformation of competent cells with pSecTag2A. Plates were spread and incubated 37C o/n.
6th August - Results from yesterday’s transformation:
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 (Main) | Yes | Yes | 100+ |
2 (Positive Control) | No | Yes | 100+ |
3 (Negative Control) | Yes | No | 15 |
This indicates that there is still an issue with the ampicillin, possibly a problem with the stock powder used. A final test with both old and new ampicillin was carried out and compared without plasmid insertion.
7th August -
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
1 Old Ampicillin | Yes | Yes | 100+ |
2 New Ampicillin v2 | No | Yes | 100+ |
3 Positive Control | Yes | No | 15 |
Results indicated that the ampicillin source may not have been fully functional, therefore a new source of amp powder was located and amp was remade.
8th August - Preparation of 4x Amp plates and 4x no drug plates for storage in the fridge.
9th August - Meeting with Darren Nesbeth, requirements for upcoming weeks: create glycerol stocks of both pSecTag2A and pSB1C3, purify and extract pure DNA for pSecTag2A and pSB1C3.
Mammalian Lab6th August - Checked HeLa cells. Cells are growing slow, left to grow for a few more days
8th August - HeLa cells are about 30% confluent. Changed media.
Week 11
Bacterial Lab
12th August - Chloramphenicol was produced and stored at -20C. In order to produce glycerol stocks, pSecTag2A and pSB1C3 were inoculated with LB media in two falcon tubes each (1x drug, 1x no drug), two plates for each plasmid were also streaked (1x drug, 1x no drug). All were left to incubate at 37C o/n.
13th August - Results from the following plates:
Vial | Ampicillin Plate | Plasmid Insertion | Colony Count |
---|---|---|---|
pSecTag2A Cells | Yes | Yes | 100+ |
W3110 Cells Amp | Yes | No | 0 |
pSecTag +ve control | No | Yes | 100+ |
PSB1C3 Cells | Yes | Yes | 0 |
W3110 Cells Chlor | Yes | No | 25 |
PSB1C3 +ve control | No | Yes | 0 |
This indicated that the pSecTag2A cells are acceptable to use for glycerol stock generation and also for plasmid purification. Miniprep was performed on the two incubated Falcon tubes from yesterday.
The results concerning pSB1C3 indicated that the chloramphenicol did not work, and the glycerol stock is dead. Therefore a new glycerol stock was sought after and chloramphenicol was remade. Amp & Cmp were remade and pSB1C3 was located in the 2012 distribution kit. Amp & Cmp were both tested by producing 1x positive and 1x negative plate for each antibiotic streaked with W3110 cells ->left to incubate o/n at 37C. glycerol stocks of pSecTag2A was grown, and a miniprep was carried out. PCR could not be carried out as the primers for pSecTag2A had not arrived.
14th August - Two analytical digests of pSecTag2A with EcoR1-HF and Dpn1 were carried out.
Item A | Volume A (ul) | Item B | Volume B (ul) |
---|---|---|---|
pSecTag2A | 5 | pSecTag2A | 5 |
EcoR1-HF | 1 | Dpn1 | 1 |
Buffer 4 | 1 | Buffer 4 | 1 |
BSA | 0.5 | BSA | 0.5 |
dH20 | 2.5 | dH20 | 2.5 |
Total | 10 | Total | 10 |
The pSB1C3 glyc stock (from HQ) was from the 2012 iGEM box in MMP -20C
Told to use a ratio of 3:1 inoculum:80% glycerol when making glyc stocks
Darren's instructions:
pSB1C3
Transfer entire glycerol tube contents to 10mL LB ND in a 50mL Falcon & measure OD. Place in 37C shakee overnight. Measure OD next morning. If there is growth in the inoculum use a loop to streak onto cmp and ND plates again.
pSecTag2A
50ul of 2013 pSecTag2A glyc stock to inoculate 10mL LB Amp in a 50mL Falcon. Grow overnight and the next day generate 15x glyc stocks sing 1.5mL eppendorfs -> store in MMP -20C.
15th August - OD results for pSB1C3 LB ND inoculum
Plasmid | OD Before | OD After | ||||||
---|---|---|---|---|---|---|---|---|
PSB1C3 (LB ND) | 0.052 | 0.5 |
ul | EcoR1 single digest | Spe1 single digest | Double digest | Uncut |
---|---|---|---|---|
pSecTag2A | 5 | 5 | 5 | 5 |
EcoR1 | 1 | 0 | 1 | 0 |
Spe1 | 0 | 1 | 1 | 0 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 |
Buffer 4 | 1 | 1 | 1 | 1 |
dH20 | 2.5 | 2.5 | 1.5 | 3.5 |
Total | 10 | 10 | 10 | 10 |
[insert image of gel]
iRRE+PC+RBS and PC+RBS (containing pSB1C3, taken from 2012 iGEM boxes) were plated onto cmp plates -> incubated o/n @37C
Two falcons for each stocks, one containing 5ul and the other 15ul, both were inoculated in 2ml LB+2ml cmp -> incushaker o/n
16th August - Falcons were retrieved:
RESULTS
Inoculum:
Falcon contents (+LB+Amp) ul | Colony growth | Absorbance |
---|---|---|
IRRE+PC+RBS (15ul) | Yes | 0.8 |
IRRE+PC+RBS (5ul) | Yes | 0.7 |
PC+RBS (15ul)/td> | No | 0.06 |
PC+RBS (5ul) | No | 0.08 |
Plates
Plate contents (+LB+Amp) ul | Colony growth | Absorbance |
---|---|---|
IRRE+PC+RBS | Yes | 100+ |
IRRE+PC+RBS | No | 100+ |
PC+RBS/td> | Yes | 0 |
PC+RBS | No | 20 |
The PC+RBS plates & falcons were discarded
IRRE+PC+RBS (15ul inoculum) -> underwent miniprep
IRRE+PC+RBS (5ul inoculum) -> 4x glycerols were made: 500ul culture + 166ul 80% glycerol, stored in MMP -20C iGEM 2013 box.
pSB1C3 Gel sd: EcoR1 & Pst1 + dd
Item (ul) | EcoR1 | Pst1 | Double digest | Uncut |
---|---|---|---|---|
pSB1C3 | 5 | 5 | 5 | 5 |
EcoR1 | 1 | 0 | 1 | 0 |
Pst1 | 0 | 1 | 1 | 0 |
Buffer 3 | 1 | 1 | 1 | 1 |
BSA | 0.5 | 0.5 | 0.5 | 0.5 |
dH20 | 2.5 | 2.5 | 1.5 | 4.5 |
Total | 10 | 10 | 10 | 10 |
3ul loading dye to each solution
Lanes:
2 - Hyperladder, 4 - EcoRI, 5 - PstI, 6 - dd, 7 - Uncut
Results from gel: 1kb DNA ladder showed on gel. No bands for EcoR1, Pst1, DD and Uncut plasmid were seen - indicating that no DNA was present -> possibly due to problems with the miniprep.
Therefore from the iGEM 2012 plate 5 (distribution kit) well 23.O, DNA containing BBa_j04450 (colonies are clearly red in colour - RFP) in pSB1C3 was located. The dried DNA was resuspended in 10ul dH2O, left to sit for 5 minutes, then transformed into competent cells -> streaked onto plates and left to incubate o/n @30C over the weekend.
Mammalian Lab
12th August - HeLa cells have confluency of about 60%. Passaged the HeLa cells and ‘backup’ HeLa cells. We set up two 6-well plates at 0.25 x 10^6 cells/ml in preparation for Zeocin kill curve. Cell count involved (0.45 cells/ml).
13th August -
Prepared media with various concentrations of zeocin for each of 6 wells.
Concentration of zeocin (µg/ml) | Volume of zeocin (ml) | Volume of DMEM + 10% FBS + 2 mM L-Glu (ml) |
---|---|---|
0 | 0 | 30.0 |
50 | 15 | 30.0 |
100 | 30 | 30.0 |
250 | 75 | 29.9 |
500 | 150 | 29.9 |
1000 | 300 | 29.7 |
T75 flasks: - 90% confluency T25 flasks - 30% confluency 6-well plates - 30% confluency
14th August - T25 flasks 90% confluency
Disc 1 Disc 2
Disc 1 | Disc 2 | Concentration of zeocin (µg/ml) | Confluency (%) | Cell Appearance | Comment | Confluency (%) | Cell Appearance | Comment |
---|---|---|---|---|---|---|
0 | 65 | Healthy | Healthy, few swell | 65 | Healthy | minimal floaters |
50 | 40 | Healthy, few swell | few floaters | 65 | occasional swell | moderate floaters |
100 | 40 | Half/moderate swell | Moderate floaters, minor infection | 50 | 40% swelling | minimal floaters |
250 | 25 | most/moderate swell | many floaters | 60 | moderate/severe swelling | many floaters |
500 | 40 | most clumps dead/ swell | many floaters, possibly infection | 65 | severe swelling all over | large number of floaters |
1000 | 45 | very severe death/ swell | many floaters, minor infection | 60 | severe swelling all over | moderate number of floaters |
The two T25 flasks with revived HeLa have confluency of about 90%; T75 flask with ‘backup’ cells 70% confluent. The T25 flasks are discarded.
16th August -
Disc 1 | Disc 2 | Concentration of zeocin (µg/ml) | Confluency (%) | Cell Appearance | Floaters | Confluency (%) | Cell Appearance | Floaters |
---|---|---|---|---|---|---|
0 | 90 | Healthy | Minimal | 95 | Healthy | minimal |
50 | 80 | minor swell | moderate | 90 | minor swell | moderate |
100 | 65 | Minor swell, minor death | Many | 75 | Minor swell, minor death | moderate |
250 | 50 | severe swelling, death | many | 50 | severe swelling, death | many |
500 | 60 | severe swelling, death | many | 50 | severe swelling, death | many |
1000 | 40 | severe swelling, death | many | 50 | severe swelling, death | many |
Split and passaged stock HeLa into 2 flasks
17th August - Passaged HeLa cells
Week 12
Bacterial Lab
19th August - pSB1C3 streaked plates (well 23.O) were retrieved from 30C incubator. Colony growth visible indicating successful transformation. Colonies were expected to be red in colour, however this was not observed.
Plate (competent cell vial) | Colony Count |
---|---|
10 | 50+ |
20 | 50+ |
A single colony from each plate was then inoculated in LB in order to later prepare glycerol stocks.
iGEM 2012 boxes were searched for pSB1C3. Transformation was carried out and then selective plates were spread, incubated o/n @37C.
Innoculations of the following were carried out in 2mL LB:
· IRRE+PC+RBS (pSB1C3) (2x in 2mL LB) -> 2ul and 5ul -> incu-shaker
· RFP+pSB1C3 (scooped from plates) (2x in 2mL LB) -> incu-shaker
^ 1.5mL miniprep, 0.5mL glycerol stock for following day (20th)
20th August - Plates taken from incubation:
Competent cells transformed with plasmid: Colony growth -> cells have successfully taken up plamid.
Negative control (only competent cells): Colony growth -> Most likely problems with Chloramphenicol.
Will therefore re-make chloramphenicol
Plate (pSB1C3+) | Colony Count |
---|---|
NUC | 5 |
LAC | 20 |
LAC | 15 |
LAC2 | 10 |
CURL1 | 30 |
Control | 50 |
take from incu-shaker:
IRRE+PC+RBS ( pSB1C3) -> 4x 200ul glycerols -> glycerol box -20C mmp
RFP+PSB1C3 -> 4x 200ul glycerols/a> ->glycerol box -20C mmp
Minipreped all 4 falcons and a gel was run -> no bands were seen indicating no DNA present.
Item (ul) | IrreAcut | IrreAuc | IrreBcut | IrreBuc | RFPcut | RFPuc | Uncut | Uncut |
---|---|---|---|---|---|---|---|---|
pSB1C3 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
EcoR1 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 |
Pst1 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 |
Buffer 3 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 |
BSA | 0.5 | 0 | 0.5 | 0 | 0.5 | 0 | 0.5 | 0 |
dH20 | 1.5 | 5 | 1.5 | 5 | 1.5 | 5 | 1.5 | 5 |
Total | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
21st August - Nanodrop of pDNA
Sample | Concentration (ng/ul) | 260/280 |
---|---|---|
Irre A pDNA | -33.0 | 1.74 |
Irre b pDNA | 123.0 | 0.94 |
GFP pDNA | -52.1 | 1.63 |
LB pDNA | -28.4 | 1.34 |
10x cmp plates were made using Yanika's chloramphenicol
Meeting with Darren:
The following Biobricks ordered were brought by our supervisor to be streaked onto the relevant antibiotic plates and inoculated in antibiotic+2mL LB -> Incubate o/n @ 37C.
Biobrick | Plate (+ND) | Inoculation (+ 2mL LB) |
---|---|---|
J63008 | 1X AMP, 1X CMP | 2 mL AMP + 2 mL CMP |
K105028 | 1X AMP | 2 mL AMP |
I712004 | 1X AMP, 1X CMP | 2 mL AMP + 2 mL CMP |
K105030 | 1X AMP | 2 mL AMP |
J63008/9 | 1X AMP | 2 mL AMP |
K105027 | 1X AMP | 2 mL AMP |
K812014 | 1X AMP, 1X CMP | 2 mL AMP + 2 mL CMP |
A PCR of Zeocin was performed and a gel was subsequently ran. PCR tube:
1 - PCR zec bb F, R, 2ul template
2 - PCR zec bb F, R, 1ul template
3 - PCR zec F, R, 2ul template
4 - PCR zec F, R, 1ul template
5 - PCR negative control zec bb F, R,
6 - PCR negative control zec F, R
PCR was unsuccessful
22nd August -
Results from Biobrick streaking and inoculation:
Biobrick | Positive Control (ND) | Cmp plate | Amp plate | Falcon |
---|---|---|---|---|
J63008 | Growth | Growth | No Growth | No Growth |
K105028 | Growth | Growth | Growth | |
K105027 | Growth | Growth | Growth | |
K105030 | Growth | Growth | Growth | |
K812014 | Growth | Growth | Growth | |
J63008/9 | Growth | Growth | Growth | |
I712004 | No Growth | No Growth | No Growth | No Growth |
Glycerol stocks were therefore made from the plates that displayed colony growth.
A second attempt at zeocin PCR was performed using Phusion DNA Polymerase
1 - Zec bb F, R 2ul
2 - Zec bb F, R 1ul
3 - Zec F, R 2ul
4 - Zec F, R 1ul
5 - Negative control zec bb F, R
6 - Negative control zec F, R
A gel was run with 8ul of each of the 6 reactions. PCR was successful.
Restriction digest of the following samples:
A - K105028
B - K105027
C - K105030
D - K812014
E - J63009/8
Double Digest | Uncut | |
---|---|---|
Sample | 5 | 5 |
EcoR1 | 1 | 0 |
Pst1 | 1 | 0 |
Buffer 3 | 1 | 0 |
BSA | 0.5 | 0 |
dH2O | 1.5 | 5 |
Total | 10 | 10 |
Sample | 260/280 | ng/ul |
---|---|---|
K105028 | 2.12 | 39.0 |
K105027 | 1.99 | 47.1 |
K105030 | 2.07 | 48.8 |
K812014 | 1.96 | 113.3 |
J63008/9 | 1.95 | 59.7 |
PCR Purification
1 - zeo bb F, R 2ul template
2 - zeo bb F, R 1ul template
^ stored in iGEM 2013 box
Preparative digest of K812014 (pSB1C3)
Sample | 5ug |
---|---|
E | 7 |
P | 7 |
B3 | 10 |
BSA | 2 |
dH2O | 30 |
Total | 100 |
5ug sample = 44ul of 113.3 ng/ul
CMV PCR
Primers used: 2s + 6FW, 2s + bbRE
23rd August -
Gel was ran with (lane):
(3) PCR purified zeo bb FR + 2ul template
(4) PCR purified zeo bb FR + 1ul template
(6) bwf + bb RE + 1ul template
(7) bwf + bb RE + 2ul template
(8) bwf + bb RE
Samples in lanes 3 & 4 failed, therefore a nanodrop was recorded:
Sample | 260/280 | ng/ul |
---|---|---|
PCR purified zeo bb FR + 2ul template | 1.66 | 55.6 |
PCR purified zeo bb FR + 1ul template | 1.82 | 17.2 |
Mammalian Lab
19th August - Viable cell count data for HeLa growth curve, Day 1 : 0.1 x 10^-6 viable cells per mL
Disc 1 Disc 2
Disc 1 | Disc 2 | Concentration of zeocin (µg/ml) | Confluency (%) | Cell Appearance | Floaters | Confluency (%) | Cell Appearance | Floaters |
---|---|---|---|---|---|---|
0 | 100 | Over confluent | Moderate | 100 | Over confluent | moderate |
50 | 50 | Moderate swelling and death | Many | 80 | Moderate swelling and death | Many |
100 | 20 | severe swelling, death | Many | 20 | severe swelling, death | many |
250 | 0 | - | many | 0 | - | many |
500 | 0 | - | many | 0 | - | many |
1000 | 0 | - | many | 0 | - | many |
Split stock HeLa cells
20th August - Viable cell count data for HeLa growth curve, Day 1 : [0.17, 0.068 (anomaly), 0.23] x 10^-6 viable cells per ml
Disc 1 Disc 2
Disc 1 | Disc 2 | Concentration of zeocin (µg/ml) | Confluency (%) | Cell Appearance | Floaters | Confluency (%) | Cell Appearance | Floaters |
---|---|---|---|---|---|---|
0 | 100 | Over confluent and death | Moderate | 90 | Over confluent | moderate |
50 | 80 | Over confluent, swelling and death | Many | 20 | Moderate swelling and death | Many |
100 | 20 | severe swelling and death | Many | 0 | severe swelling, death | many |
250 | 0 | - | many | 0 | - | many |
500 | 0 | - | many | 0 | - | many |
1000 | 0 | - | many | 0 | - | many |
21st August - Viable cell count data for HeLa growth curve, Day 1 : [0.496, 0.244 (anomaly), 0.356] x 10^-6 viable cells per ml
22nd August - Viable cell count data for HeLa growth curve, Day 1 : [0.79, 1.12, 1.19] x 10^-6 viable cells per ml
Week 13
Bacterial Lab
27th August -
Purify zeocin CMV from zeo FB primers
Zeocin and CMV purification
Tube:
1 - CMV 1ul template bb F, R
2 - CMV 2ul template bb F, R
3 - Zeocin 2ul template zecF, R
4 - Zeocin 1ul template zecF, R
5 - Zeocin 2ul template zecbbF, R
6 - Zeocin 1ul template zecbbF, R
28th August - 3x glycerol stocks of K812014 were prepared from inoculum. 1.5mL of culture was used for miniprep.
Nanodrop of Zec and CMV was recorded:
260/280 | ng/ul | |
---|---|---|
ZEC | 1.68 | 21.1 |
CMV | 1.96 | 82.1 |
A gel was run on 100ul of prep digest K812014 + 20ul dye. 2000bp was scooped in gel extraction.
Chloramphenicol was tested at x2 and x4 concentration with 50ul cells spread on each plate, results from the next day still showed significant colony growth (100+).
29th August - Nanodrop of pSB1C3 purified from gel extract, 260/280: -25/21, ng/ul: 13.5
5ul was run on a gel, however extract of pSB1C3 was unsuccessful as no bands were visible.
PCR:
3 PCR reactions of zeocin using zec BB F, R primers were performed. First reaction: 1 ul pSecTag2A template, second 2 ul pSecTag2A and third reaction - negative control - no template.
3 PCR reactions for cmv promoter were prepared using the same variation of template volume as above. These were tested the next day and were successful. Gel photo (30/08).
Prep digest+gel+extract+purify+nanodrop+5ul gel of zeocin and CMV were carried out
Prep digest:
Sample (ul) | Zeo | CMV | Cyc100 | Cyc100 | Cyc70 | Cyc70 | Cyc28 | Cyc28 |
---|---|---|---|---|---|---|---|---|
V. Sample | 48 | 45 | 17.5 | 17.5 | 23 | 23 | 23.5 | 23.5 |
B3 | 10 | 10 | 5 | 5 | 5 | 5 | 5 | 5 |
E | 7 | 7 | 5 | 5 | 5 | |||
P | 7 | 7 | 5 | 5 | 5 | 5 | 5 | 5 |
X | 5 | 5 | ||||||
BSA | 2 | 2 | 1 | 1 | 1 | 1 | 1 | 1 |
H2O | 26 | 29 | 16.5 | 16.5 | 11 | 11 | 10.5 | 10.5 |
Total | 100 | 100 | 50 | 50 | 50 | 50 | 50 | 50 |
Glycerol stocks (x15) of J63009, K105027, K105028 and K105030 were made.
Additionally, nanodrops were recorded:
Sample | 260/280 | Absorbance | ng/ul |
---|---|---|---|
J63009 | 2.10 | 0.774 | 88.7 |
K105030 | 209.00 | 0.653 | 60 |
K105028 | 2.14 | 0.408 | 50 |
K105029 | 2.18 | 0.585 | 77.0 |
K218014 | 2.12 | 0.946 | 104.9 |
A prep digest was conducted on K812014 to extract pSB1C3.
Chloramphenicol was tested again but this time with x8 cmp (80ul cmp to 10mL LB agar). Overgrad, undergrad and Yanika chloramphenicol stocks were used and 50ul competent cells were spread -> incubate o/n @37C.
Zec and CMV nanodrops were recorded:
Sample | 260/280 | Absorbance | ng/ul |
---|---|---|---|
ZEC | 1.87 | 9.538 | 2.5 |
CMV | 2.06 | 11.048 | 25.4 |
30th August
Chloramphenicol test - plate colony growth observation and count: all 3 plates prepared using the chloramphenicol from postgrads, us and Yanika had 30+ very small colonies.
After overnight incubation, from each of the the 10 ml inoculations of K812014 (I), pSecTag2A and second (different) sample of K812014 (II), 2.5 ml were minipreped while the rest of 7.5 ml from each was used to make glycerol stocks.
Nanodrop of these minipreps: