Difference between revisions of "Team:Potsdam/notebook/lowcopy"
| Line 817: | Line 817: | ||
- Tac2: 9,2 ng/µL <br> | - Tac2: 9,2 ng/µL <br> | ||
- LacI: 12,9 ng/µL </div> | - LacI: 12,9 ng/µL </div> | ||
| + | <br> <br> | ||
| + | |||
| + | <b> Tuesday, 08/29/17 </b> | ||
| + | <br> <br> | ||
| + | <div style="text-align: justify;"> | ||
| + | <b>Colony-PCR</b> of P4 with Q24 and Q18 (see protocol and note the changes in the folder!)</div> | ||
| + | <div style="text-align: justify; margin-left:40px"> | ||
| + | - 10 colonies <br> | ||
| + | - masterplate <br> | ||
| + | - did a test gel (1 % Agarose) (actualy did two beacuse of missing marker in the first one, see lab book)<br> | ||
| + | - gel picture shows: all fragments have expected length <br> | ||
| + | - no miniprep culturs inoculated because colonies were too small -> To do tomorrow </div> | ||
| + | <div style="text-align: justify;"> | ||
| + | <b>Gibbson assembly</b> of P5 (IAA-RBP in pSB4A5) (see online protocol from NEB) | ||
| + | <b>Transformation</b> of P5 into Jm109</div> | ||
| + | <div style="text-align: justify;"> | ||
| + | - for tomorrow: inoculate miniprep cultures of positive clones on masterplate for P4, Colony-PCR and test gel for P5 </div> | ||
| + | <br> <br> | ||
| + | |||
| + | <b> Wednesday, 08/30/17 </b> | ||
| + | <br> <br> | ||
| + | <div style="text-align: justify;"> | ||
| + | <b>Colony PCR</b> of P5 (low copy plasmid, see protocol) with Q19 and Q20<br> | ||
| + | - gel picture: all clones okay<br> | ||
| + | - inoculated three precultures for minipreps tomorrow (10 ml, 37 °C shaker)<br> | ||
| + | - also inoculated three precultures of P4 for miniprep tomorrow (10 ml, 37 °C shaker)</div> | ||
<br> <br> | <br> <br> | ||
</p> | </p> | ||
Revision as of 16:34, 1 November 2017
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:
2. pSB4A5:
Colony-PCR with the SLiCE plate and at the same time preparation of the Slakowski-Assay plate
Friday, 08/18/17
Monday, 08/21/17
- 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!
- 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!
2. 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:
- 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
+ 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)
- 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
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
4. for tomorrow:+ 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
Colony-PCR with the SLiCE plate and at the same time preparation of the Slakowski-Assay plate
Friday, 08/18/17
- no colonies on pSB4A5 plate
- did q5-Q8 PCr again with 50 uL reaction following protocol (5 uL of 1:100 diluted miniprep as template)
used Q5678 protocol in Torsten Thermocycler
- gel extraction:
- did q5-Q8 PCr again with 50 uL reaction following protocol (5 uL of 1:100 diluted miniprep as template)
used Q5678 protocol in Torsten Thermocycler
- gel extraction:
fragment A: 3,7 ng/uL
fragment b: 31,4 ng/uL
fragment b: 31,4 ng/uL
- PCR Q5/Q8 again for fragment A with gradient PCR
- for Monday: program in Torsten: "Q58":
- for Monday: program in Torsten: "Q58":
- use 4 different wells/temperatures:
- well 1: 60°C; 5: 62,5°C; 7:64,5 °C; 9: 66,5°C
- elongation temperature reduced to 30 sec
- well 1: 60°C; 5: 62,5°C; 7:64,5 °C; 9: 66,5°C
- elongation temperature reduced to 30 sec
- make 50 uL reaction again,
afterwards: gel extraction and fusion PCR of dCas-A and dCas9-B
ask Fabian for better Fusion-PCR cycler program (the one from last time was unnecessarily long according to Fabian)
afterwards: gel extraction and fusion PCR of dCas-A and dCas9-B
ask Fabian for better Fusion-PCR cycler program (the one from last time was unnecessarily long according to Fabian)
Monday, 08/21/17
- gradient PCR for dCas9-A fragment
- Mastermix for 10 reactions (10 µl/ reaction):
| component | volume in µl |
|---|---|
| Q5 2xMastermix | 50 |
| primer Q5 (10µM) | 5 |
| primer Q8 (10µM) | 5 |
| water | 20 |
| template (~1ng) | 20 |
| total volume | 100 |
- template: pdCas9 from miniprep (48,3 ng/µl) -> dilution: 1:100 ~ 0,5 ng/µl
- PCR program: dcas9a_gradient (in Torsten) (also changed elongation time to 30 s)
temperatures of gradient:
60°C 60,2°C 61,5°C 62,5°C 64,5°C 65,5°C 67,8°C 68°C
- 1 % agarose gel -> 25 min, 140 V
- gel picture:
- PCR program: dcas9a_gradient (in Torsten) (also changed elongation time to 30 s)
temperatures of gradient:
60°C 60,2°C 61,5°C 62,5°C 64,5°C 65,5°C 67,8°C 68°C
- 1 % agarose gel -> 25 min, 140 V
- gel picture:
- used annealing temperature of 60,2°C (B) for preparative PCR (50 µl / reaction):
- two different reactions with different template amounts:
- reaction 1: 0,5 ng
- reaction 2: 5 ng
- reaction 2: 5 ng
| component | volume in µl |
|---|---|
| Q5 2xMastermix | 25 |
| primer Q5 (10µM) | 2,5 |
| primer Q8 (10µM) | 2,5 |
| water reaction 1/2 | 19/10 |
| template reaction 1/2) | 1/10 |
| total volume | 50 |
- 1 % agarose gel -> 30 min, 120 V
- excision of gel bands from reaction 1 and 2 (1,3 kb) -> put in one tube
- stored at 4 °C over night
Tuesday, 08/22/17
Wednesday, 08/23/17
- excision of gel bands from reaction 1 and 2 (1,3 kb) -> put in one tube
- stored at 4 °C over night
Tuesday, 08/22/17
- Gel clean-up of dCas9-A (Elution with 50 µl NE-Buffer) -> c = 17,6 ng/µl
- Fusion PCR
- Fusion PCR
- changes in the PCR Program (in Torsten):
- elongation time in the second cycle: 3 min 18 s (30 s/kb * 6,6 kb (size of the fusion product) = 198 s)
- finale elongation time after the second cycle: 10 min
- finale elongation time after the second cycle: 10 min
- for the first part of the fusion PCR:
| component | volume in µl |
|---|---|
| Q5 2xMastermix | 12.5 |
| dCas9-A (50 ng/µl) | 2.8 |
| dCas9-B (50 ng/µl) | 1.6 |
| water | 8.1 |
| template reaction 1/2) | 1/10 |
| total volume | 25 |
at the "hold-step" in PCR program added the primer mix (5 µl) and continued the PCR:
| component | volume in µl |
|---|---|
| Q5 2xMastermix | 2.5 |
| dCas9-A (50 ng/µl) | 1.25 |
| dCas9-B (50 ng/µl) | 1.25 |
| total volume | 5 |
- DpnI digestion after the fusion PCR:
- added 1 µl DpnI to the PCR product
- incubated 30 min at 37°C
- incubated 30 min at 37°C
stored digested PCR product at -20 °C
Wednesday, 08/23/17
- gel extraction of fusion-PCR product (1 % agarose, next time use 0, 7%; 100V. 55 min)
- elution in 45 uL NE-Buffer (25 + 20), concentration: 3,3 ng/µL
- not enough --> PCR amplification with Q5/Q6 with standard 50 µL reaction (0,5 µL Plasmid template)
- program "dCas9 after fusion in PeqSAR mixer, Eppi: "PCR fusion", 35 cycles
- elution in 45 uL NE-Buffer (25 + 20), concentration: 3,3 ng/µL
- not enough --> PCR amplification with Q5/Q6 with standard 50 µL reaction (0,5 µL Plasmid template)
- program "dCas9 after fusion in PeqSAR mixer, Eppi: "PCR fusion", 35 cycles
| temperature | time |
|---|---|
| 98 | 30 |
| 98 | 10 |
| 64 | 30 |
| 72 | 3:21 min |
| 72 | 2 min |
- into -20, tomorrow test gel run
- Primer for amplification (Q25-Q30) resuspended to 100µM and separately diluted to 10 µM
- IDT-fragments were diluted 1:10 separately
- PCR for amplification of PCR fragments:
- pipetted normal 50 µL reaction (1 µL of diluted fragment)
- PCR reaction ("idt" in Torsten): 35 cycles
- Primer for amplification (Q25-Q30) resuspended to 100µM and separately diluted to 10 µM
- IDT-fragments were diluted 1:10 separately
- PCR for amplification of PCR fragments:
- pipetted normal 50 µL reaction (1 µL of diluted fragment)
- PCR reaction ("idt" in Torsten): 35 cycles
| temperature | time |
|---|---|
| 98 | 30 |
| 98 | 10 |
| gradient 58-71 | 20 |
| 72 | 60 |
| 72 | 2 min |
- PP7 and MS2 at 71 °C, LacI at 58 °C
- put into -20 and tomorrow test gel run
Thursday, 08/24/17
Friday, 08/25/17
Monday, 08/28/17
Tuesday, 08/29/17
Wednesday, 08/30/17
- put into -20 and tomorrow test gel run
Thursday, 08/24/17
- Fusion PCR: purifaction gel electrophoresis
- gel electrophoresis:
- 50 µl DNA
- 10 µl Loading Dye
- 0,7 % Agarose gel
- 6,6kb fragment
- 1h with 80 V
- 10 µl Loading Dye
- 0,7 % Agarose gel
- 6,6kb fragment
- 1h with 80 V
- purifaction with the Promega Purification Kit
- final concentration: 45,7 ng/µL
- test gel of PCR with IDT fragments of yesterday:
IAAH-PP7 - IAAM-MS2 - LacI - TaC1 - TaC2 - TaC3 - Ddx4-YFP
- final concentration: 45,7 ng/µL
- test gel of PCR with IDT fragments of yesterday:
IAAH-PP7 - IAAM-MS2 - LacI - TaC1 - TaC2 - TaC3 - Ddx4-YFP
- only IAAH-PP7 and TaC3 bands are okay
- gel purification: PP7: 40,7 ng/µL
- TaC3: 27,3 ng/µL
- second PCR with other fragments, used three different temperatures for annealling for every fragment:
- gel purification: PP7: 40,7 ng/µL
- TaC3: 27,3 ng/µL
- second PCR with other fragments, used three different temperatures for annealling for every fragment:
| fragment | 1st TM (well) | 2nd TM | 3rd TM |
|---|---|---|---|
| MS2 | 68,3 (9) | 69,7 (10) | 71 (12) |
| LacI | 56 (1) | 57,3 (3) | 60,7 (5) |
| TaC1 | 66,3 (8) | 68,3 (9) | 69,7 (10) |
| TaC2 | 68,3 (9) | 69,7 (10) | 71 (12) |
| YFP | 62,6 (6) | 64,4 (7) | 66,3 (8) |
- Tip from Fabian: next time more gradient Temperature distance!
- fragments from left to right (Temperatures are alaso from left to right) : MS2, LacI, TaC1, TaC2, YFP
2017-08-24 15hr 44min-1.jpg
- fragments from left to right (Temperatures are alaso from left to right) : MS2, LacI, TaC1, TaC2, YFP
- MS2 / 9 band correct
- YFP / 6 and 7 bands are correct
- LacI no bands, TaC1 almost no bands, TaC2 bands incorrect
- gel run of MS2/9 and YFP/ 6,7 and put cut gel slices in the fridge for extraction tomorrow
- test gel of 4 uL (10 ng/uL) of IDT-fragments: LacI, TaC1, TaC2 with 6 uL H2O und 2 uL Dye to see if there's something wrong with the IDT fragments
- YFP / 6 and 7 bands are correct
- LacI no bands, TaC1 almost no bands, TaC2 bands incorrect
- gel run of MS2/9 and YFP/ 6,7 and put cut gel slices in the fridge for extraction tomorrow
- test gel of 4 uL (10 ng/uL) of IDT-fragments: LacI, TaC1, TaC2 with 6 uL H2O und 2 uL Dye to see if there's something wrong with the IDT fragments
- TaC1 and YFP show correct bands
- TaC2 correct and wrong band (maybe low concentration?) --> Nanodrop result: 11,4 ng/uL; problem that DNA concentration of desired
DNA is too low for restriction?
- TaC2 correct and wrong band (maybe low concentration?) --> Nanodrop result: 11,4 ng/uL; problem that DNA concentration of desired
DNA is too low for restriction?
Friday, 08/25/17
- Gel and PCR clean up (Promega kit)
- MS2: 240mg gel; final concentration: 16,6 ng/µL
- YEP 6: 250mg gel; final concentration: 19,6 ng/µL
- YEP 7: 200mg gel; final concentration: 10,9 ng/µL
- YEP 6: 250mg gel; final concentration: 19,6 ng/µL
- YEP 7: 200mg gel; final concentration: 10,9 ng/µL
Gibbson Assembly "NEBuilder® HiFi DNA Assembly Master Mix"
https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
https://www.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol
| component | volume in µl | size in bp | concentration |
|---|---|---|---|
| Vector: psB4A5 (IG_C_P1_linearized) | 2,75 | 3395 | |
| Insert 1: IAAM-MS2 | 6,1 | 2200 | 10,6 ng/µl |
| Insert 2: IAAH-PP7 | 1,3 | 1800 | 40,7 ng/µl |
| Master Mix | 10,15ul |
Trafo
https://www.neb.com/protocols/2015/02/17/nebuilder-hifi-dna-assembly-chemical-transformation-protocol-e2621
https://www.neb.com/protocols/2015/02/17/nebuilder-hifi-dna-assembly-chemical-transformation-protocol-e2621
- Gibbson Assembly (5 µl) + competen cells (C2987)
- plate cells: 100µl and 200µl
- plate cells: 100µl and 200µl
Monday, 08/28/17
- Gibson Assembly worked, we have colonies but they are useless (see below)
- colony PCR following the protocol (beware that the protocol has been updated in the lab),
annealing Temperature: 52°C wiht Q24 and Q18 and 10 colonies
- negative control: IG_C_P1 miniprep
- negative/Gibson 1-10 and TaC3
- colony PCR following the protocol (beware that the protocol has been updated in the lab),
annealing Temperature: 52°C wiht Q24 and Q18 and 10 colonies
- negative control: IG_C_P1 miniprep
- negative/Gibson 1-10 and TaC3
- negative results for all reactions except the last onw which is just the gel purified TaC3
- excpept it isnt because the band cleearlyc shwows a sifferent size and is more likely to be IAAAH-PP7 (a suspicion of Sarah who wasn't sure if the tubes were swapped) which explains the negative results in the Gibson assembly
- Gibson assembly was done again withcorrect fragments:
- excpept it isnt because the band cleearlyc shwows a sifferent size and is more likely to be IAAAH-PP7 (a suspicion of Sarah who wasn't sure if the tubes were swapped) which explains the negative results in the Gibson assembly
- Gibson assembly was done again withcorrect fragments:
- pSB4A5 miniprep: 2,75 uL (18 ng/µL)
- IAAM-MS2 6,1 uL (10,6 ng/µL)
- IAAH-PP7 1,9 uL (27,3 ng/µL)
- MasterMix 10,77 µL (fragments to Mastermix : 1:1)
- IAAM-MS2 6,1 uL (10,6 ng/µL)
- IAAH-PP7 1,9 uL (27,3 ng/µL)
- MasterMix 10,77 µL (fragments to Mastermix : 1:1)
- two Trafos were done with already used competent cells and new competent cells (always use the cells we got from - the Gibson Kit for Gibson assembly!) and plated with 200 uL and centrifuged
- repeated PCR to amplify IDT-fragments with TaC1, TaC2 and LacI (always as 50 uL reactions):
- repeated PCR to amplify IDT-fragments with TaC1, TaC2 and LacI (always as 50 uL reactions):
- 3 different concentrations and temperatures for each fragment:
| temperature in °C/ concentration | 0,1 ng | 1 ng | 10 ng |
|---|---|---|---|
| 60 | 1 | 4 | 7 |
| 68 | 3 | 6 | 9 |
from left to right: Marker- TaC1/ 1-9 - one empty lane - TaC2/1-4 - Marker
Marker - TaC2/5-9 - LacI/ 1-9 (but not 5!) - Marker - 6LacI/5
low concentrations worked best, results at Tac1/1, TaC2/3 and LacI/1 were best and gel purified
- Tac1: 5,8 ng/µL
- Tac2: 9,2 ng/µL
- LacI: 12,9 ng/µL
- Tac2: 9,2 ng/µL
- LacI: 12,9 ng/µL
Tuesday, 08/29/17
Colony-PCR of P4 with Q24 and Q18 (see protocol and note the changes in the folder!)
- 10 colonies
- masterplate
- did a test gel (1 % Agarose) (actualy did two beacuse of missing marker in the first one, see lab book)
- gel picture shows: all fragments have expected length
- no miniprep culturs inoculated because colonies were too small -> To do tomorrow
- masterplate
- did a test gel (1 % Agarose) (actualy did two beacuse of missing marker in the first one, see lab book)
- gel picture shows: all fragments have expected length
- no miniprep culturs inoculated because colonies were too small -> To do tomorrow
Gibbson assembly of P5 (IAA-RBP in pSB4A5) (see online protocol from NEB)
Transformation of P5 into Jm109
- for tomorrow: inoculate miniprep cultures of positive clones on masterplate for P4, Colony-PCR and test gel for P5
Wednesday, 08/30/17
Colony PCR of P5 (low copy plasmid, see protocol) with Q19 and Q20
- gel picture: all clones okay
- inoculated three precultures for minipreps tomorrow (10 ml, 37 °C shaker)
- also inoculated three precultures of P4 for miniprep tomorrow (10 ml, 37 °C shaker)
- gel picture: all clones okay
- inoculated three precultures for minipreps tomorrow (10 ml, 37 °C shaker)
- also inoculated three precultures of P4 for miniprep tomorrow (10 ml, 37 °C shaker)
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