Lab book
We splitted our labbook into the following parts:
Friday, 07/21/17
- took pSB4A5 backbone out of DNA distribution Kit and transformed into E.coli
(rest from Kit was put in -20 °C)
Tuesday, 07/25/17
- Inoculated LB media with psB4A5
Wednesday, 07/26/17
- Miniprep with Promega “Plus SV Miniprep DNA Purification System”
Thursday, 08/10/17
- glycerol stock (30%) of IG_C_P1_G (pSB4A5)
- prepared 10 mL preculture with pdCas9-Bacteria E.coli from plate from Fabian for Miniprep
Monday, 08/14/17
- primer resuspended to 100 uM (except Q8):
- diluted all primers to 10 uM in separate aliquotes
- prepareed 3 separate PCRs with primer pairs:
Q5/Q8 (Eppi A), Q6/Q7 (Eppi B), Q15/Q16 (Eppi c)
- pipetting scheme:
- after PCR: gel electrophoresis as follows:
- 2.5 uL DNA + 2uL Loading Dye + 7.5 uL (total 12 uL)
- ran gel at 120 V for 40 minutes
gel picture:
- order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again
- dCas9 fragments look good and can be worked with tomorrow
Tuesday, 08/15/17
1. DpnI digestion of dCas9 fragments A + B
- Duration: 1 hour
IMPORTANT: Fabian told me you can just add about 1 uL DpnI to the reaction-mix
2. Gel run of dCas9 A + B
3. Linearization PCR of psB4A5 (second atempt) via gradient PCR
(rest from Kit was put in -20 °C)
Tuesday, 07/25/17
- Inoculated LB media with psB4A5
Wednesday, 07/26/17
- Miniprep with Promega “Plus SV Miniprep DNA Purification System”
- 2 approaches with psB4A5
- final concentration:
- final concentration:
- approach 1: 30 ng/ul
- approach 2: 40 ng/ul
- approach 2: 40 ng/ul
Thursday, 08/10/17
- glycerol stock (30%) of IG_C_P1_G (pSB4A5)
- prepared 10 mL preculture with pdCas9-Bacteria E.coli from plate from Fabian for Miniprep
Monday, 08/14/17
- primer resuspended to 100 uM (except Q8):
primer | volume in µl |
---|---|
Q5 | 354 |
Q6 | 957 |
Q7 | 749 |
Q8 | 657 (83,1 uM) |
Q15 | 956 |
Q16 | 1455 |
Q17 | 1693 |
Q18 | 1440 |
- prepareed 3 separate PCRs with primer pairs:
Q5/Q8 (Eppi A), Q6/Q7 (Eppi B), Q15/Q16 (Eppi c)
- pipetting scheme:
Q5 2X MasterMix: 5 uL
10 uM Primer: each 0,5 uL
Template: 1 uL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 uL
total: 10 uL
- cycling conditions (prog01 in Epepndorf Mastercycler nexus gradient (Torsten):10 uM Primer: each 0,5 uL
Template: 1 uL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 uL
total: 10 uL
temperature | time in sec |
---|---|
98 °C | 30 |
98 °C | 10 |
64 °C | 20 |
72 °C | 110 |
72 °C | 120 |
- 2.5 uL DNA + 2uL Loading Dye + 7.5 uL (total 12 uL)
- ran gel at 120 V for 40 minutes
gel picture:
- order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again
- dCas9 fragments look good and can be worked with tomorrow
Tuesday, 08/15/17
1. DpnI digestion of dCas9 fragments A + B
component | volume in µl |
---|---|
DpnI | 2,6 |
DNA | 6,5 |
cutsmart-Buffer | 5,0 |
H2O | 35,9 |
final volume | 50 |
IMPORTANT: Fabian told me you can just add about 1 uL DpnI to the reaction-mix
2. Gel run of dCas9 A + B
- let it run on gel for purification
- 140 V a 40 min
- cut out was done by Natalie and Pauline
- 140 V a 40 min
- cut out was done by Natalie and Pauline
3. Linearization PCR of psB4A5 (second atempt) via gradient PCR
- made new PCR using Q15/Q16 primers and psB4A5 plasmid fpr linearization
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)
- annealing temperatures: A = 60°C/B = 64,5°C/C = 68°C
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)
component | 1 reaction: volume in µl |
10 reaction MM: volume in µl |
---|---|---|
HiQ | 5 | 50 |
primer | 1,25 | 12,5 |
DNA | variable (1 µl) | variable |
H2O | 17,5 | 175 |
final volume | 25 | 250 |
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:
0,1 A | 0,1 B | 0,1 C |
1 A | 1 B | 1 C |
10 A | 10 B | 10 C |
Main sponsors: