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 µL DNA + 2µL Loading Dye + 7.5 µL (total 12 µL)
- 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 µL 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/µl
- approach 2: 40 ng/µl
- approach 2: 40 ng/µl
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 µL
10 uM Primer: each 0,5 µL
Template: 1 µL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 µL
total: 10 µL
- cycling conditions (prog01 in Epepndorf Mastercycler nexus gradient (Torsten):10 uM Primer: each 0,5 µL
Template: 1 µL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 µL
total: 10 µL
temperature | time in sec |
---|---|
98 °C | 30 |
98 °C | 10 |
64 °C | 20 |
72 °C | 110 |
72 °C | 120 |
- 2.5 µL DNA + 2µL Loading Dye + 7.5 µL (total 12 µL)
- 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 µL 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:
Wednesday, 08/16/17
1. Gel run of psB4A5 gradient PCR (A,B,C,0.1,1,10)
IMPORTANT: Fusion PCR done like team munich!
https://static.igem.org/mediawiki/2014/c/c9/LMU_Munich14_FusionPCR.pdf
Thursday, 08/17/17
1. dCas9:
- 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 |
Wednesday, 08/16/17
1. Gel run of psB4A5 gradient PCR (A,B,C,0.1,1,10)
- run at 120 V a 40 min -> only 2 bands at well 1A
- run at 120 V another 30 min -> lost DNA from gel
- 1A (60°C/1:100 DNA) might have worked so I did a PCR purification for this one
2. Gel purification of dCAs9 A + B fragments and 1A-psB4A5 linearized plasmid - run at 120 V another 30 min -> lost DNA from gel
- 1A (60°C/1:100 DNA) might have worked so I did a PCR purification for this one
- used Promega PCR/Gel purification kit: final concentration pSB4A5 18 ng/µL
https://www.promega.de/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/
IMPORTANT: We use AE-buffer for elution in the last step (50 µL) instead of water!
3. Fusion PCR of dCAs9 fragments A + B https://www.promega.de/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/
IMPORTANT: We use AE-buffer for elution in the last step (50 µL) instead of water!
- Primer used: Q5 + Q6
component | volume in µl |
---|---|
Q5-MM | 12,5 |
DNA | 2(1 pro fragment) |
H2O | 10,5 |
final volume | 25 |
IMPORTANT: Fusion PCR done like team munich!
https://static.igem.org/mediawiki/2014/c/c9/LMU_Munich14_FusionPCR.pdf
- first made a PCR mix of 25 µL without primers
- then run mix in thermocycler with programm (fusion pcr)
- in the middle there is a hold step in the programm
- you then add the primers mixed with buffer (5 µL total volume) for an endvolume of 30 µL
- IG_C_Q19/20 and Q24 resuspended auf 100 µM and diluted to 10 µM
- then run mix in thermocycler with programm (fusion pcr)
- in the middle there is a hold step in the programm
- you then add the primers mixed with buffer (5 µL total volume) for an endvolume of 30 µL
- IG_C_Q19/20 and Q24 resuspended auf 100 µM and diluted to 10 µM
Thursday, 08/17/17
1. dCas9:
- DpnI- digested Fusion-PCR product (1 µL in PCR-tube for 1h at 37 °C)
- ran 0,7 % agarose gel --> negative result
- gel picture:
- ran 0,7 % agarose gel --> negative result
- gel picture:
- possible reasons: not enough DNA in PCRPCR cleanup was done and not gel extraction, wrong PCR protocol
- PCR with Q5-Q8 was done again (see above for protocol)
- no test gel but preperative gel and gel extraction with Promega protocol
- dCas9-A fragment: 2,8 ng/µL, dCas9-B fragment: 3,3 ng/µL
- not enough DNA again for PCR, tomorrow run PCR with 50 µL reaction tomorrow!
pSB4A5:
DpnI-digestion:
1 µl added to the PCR mix and incubated 1 h at 37°C
SLiCE reaction of pSB4A5 with IAAM-MS2 + IAAH-PP7:
because of to low DNA concentration we needed to increase the concentration by drying the DNA
for this reaction we take:
2,8 µl backbone (50 ng, 3395 bp, 18 ng/µl)
+ 32,2 µl IAAM-MS2 (1:10 ratio, 2188 bp, 10 ng/µl)
+ 26,4 µl IAAH-PP7 (1:10 ratio, 1790 bp, 10 ng/µl)
all in one eppi + 38,6 µl ddwater to get 100 µl
to the 100 µl we added:
+ 1/10 of original volume (100µl) 3 M Sodium acetate, pH 5,2 --> 10 µl
+ 2 x original volume 100% ethanol (needs to be cooled down for 20 min at -20°C) --> 200 µl
mix thorougly
put 30 min to -20°C (in this time cool down centrifuge to 4°C)
centrifuge 10 min with maiximal speed at 4°C
discard supernatant with a pipette, BE CAREFUL!!!, pellet is maybe (in most cases, because we don't have much DNA) nit visible, pay attenteion on the orinetation of the Eppis in the centrifuge (lids to the outside) and go with the pipette onto this side of the Eppi, where the pellet DON'T is, DON'T TOUCH THE PELLET!!!
add 300 µl 70% ethanol (needs to be cooled down for at least 20 min at -20°C)
centrifuge 10 min with maiximal speed at 4°C
again CAREFULLY discard supernatant with a pipette
make a short spin 10-30 sec (Short-Button) and take carefully additional supernatant with a pipette
put Eppi open lid for 10 min to 37°C
add SLiCE reaction directly to this Eppi (NO RESUSPENSION BEFORE)
SLiCE reaction:
Eppi with dried backbone, IAAM-MS2 and IAAH-PP7
+ 1 µl 10 x SLiCE buffer
+ 1 µl PPY SLiCE extract
+ 8 µl ddwater
--> 10 µl SLiCE reaction
incubate 1 h at 37°C, no shaking (flip a few times to the Eppi to resuspend the DNA)
Transformation in E.coli clls with all the 10 µl SLiCE reaction in the same Eppi like the transformation protocol, no changes from protocol
200 µl to Ampicillin-LB-agar plates
and centrifuge rest of the culture, discard most of the supernatant, resuspend in the rest supernatant and put this resuspenden rest also to an Ampicillin-LB-agar plate
plates over night to 37°C
for tomorrow: Colony-PCR with the SLiCE plate and at the same time preparation of the Slakowski-Assay plate
- PCR with Q5-Q8 was done again (see above for protocol)
- no test gel but preperative gel and gel extraction with Promega protocol
- dCas9-A fragment: 2,8 ng/µL, dCas9-B fragment: 3,3 ng/µL
- not enough DNA again for PCR, tomorrow run PCR with 50 µL reaction tomorrow!
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