Difference between revisions of "Team:Munich/engineering2.html"
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| − | <td>pRED/ET engineering | + | <td> |
| + | <a href="https://static.igem.org/mediawiki/2018/b/bf/T--Munich--pRED_ET-Ecoli_Gene_Deletion_Kit.pdf.pdf" target="_blank">pRED/ET engineering</a> | ||
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<td>Protocol:</td> | <td>Protocol:</td> | ||
| − | <td>pRED/ET engineering | + | <td><a href="https://static.igem.org/mediawiki/2018/b/bf/T--Munich--pRED_ET-Ecoli_Gene_Deletion_Kit.pdf.pdf" target="_blank">pRED/ET engineering</a></td> |
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Revision as of 23:28, 16 October 2018
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 |
| 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 |
| 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 |
