Team:Munich/engineering2.html
Transforming E. Coli Rosetta and MG1655 for pRED/ET engineering
2018/08/28| Participants: | Dominic Schwarz |
| Protocol: | Electrocompetent transformation |
| Notes: | Genetic engineering is planned in E. Coli Rosetta, MG1655 is E.Coli WT as backup. |
| Results: | colonies only on E. Coli MG1655 plate; No colonies on E. Coli Rosetta |
pRED/ET genome engineering of delta msb-B and delta recBCD strains
2018/08/29| Participants: | Dominic Schwarz |
| Protocol: | pRED/ET engineering protocol |
| Notes: | the template for the resistance cassette for deletions was taken from a plasmid containing mRFP |
| Results: | red colonies. Elena (Advisor) and Dominic decided to repeat the experiment but to do a dpnI digest before to get rid of the template DNA. |
Transforming E. Coli DH5a to find a reason for the contamination
2018/08/30| Participants: | Dominic Schwarz |
| Protocol: | Electrocompetent transformation |
| Notes: | CAP_recBCD, CAP_msbb, psb1c3_mrfp in Dh5a |
| Results: | red colonies on all plates -> mRFP contamination |
Creating a selection cassette from pSB1C3
2018/08/30| Participants: | Dominic Schwarz |
| Protocol: | Restrition digest, PCR purification |
| Notes: | DpnI |
| Results: | CAP_recBCD 18ng/ul CAP_msbB 12ng/ul |
pRED/ET genome engineering of delta msb-B and delta recBCD strains
2018/09/01| Participants: | Dominic Schwarz |
| Protocol: | pRED/ET engineering protocol |
| Notes: | the template for the resistance cassette for deletions was taken from a plasmid containing mRFP |
| Results: | colonies on both plates |
Verifying deletion strains of E. Coli MG1655
2018/09/05| Participants: | Dominic Schwarz |
| Protocol: | Colony PCR, Agarose gel |
| Notes: | Primers: VF2, geno_msb-B_rv, geno_recBCD_rv;
Ta: 48°C
t= 1kb/min RecBCD: 1,2,3,4,5 msbB: 35,36,37,38,39 expected: RecBCD 443bp Expected: msb-B 574bp |
| Results: | all 5 picked colonies were positive for the insertion of the selection cassette PIC |
