Team:Munich/chibiobrick2.html
Forming oligodimers from single-strand DNA chi6 sequences
2018/07/31| Participants: | Domi |
| Protocol: | Oligodimerization |
Assembling pSB1C3_Chi6 with A3 Assembly
2018/08/01| Participants: | Domi |
| Protocol: | Restriction digest, PCR purify, Gibson assembly, chem.trafo |
| Notes: | Restriction digest with XbaI, SpeI-HF |
| Results: | no colonies |
Redo: Assembling pSB1C3_Chi6 with Ligation
2018/08/02 - 2018/08/06| Participants: | Enikö |
| Protocol: | PCR, PCR purify, gibson assembly, ligation, chem trafo, mini-prep, agarose gel |
| Notes: | We performed linear amplification of Chi6 single stranded DNA because assembly failed before. gibson assembly and ligation was performed in parallel Primer: BBS-PstI-rv |
| Results: | Colonies were obtained but consequent test-pcr showed no correct bands on the gel. The colonies had to be contaminants. Eni suspected errors during the PCR reaction. |
Redo: Assembling pSB1C3_Chi6 with Ligation
2018/08/07| Participants: | Enikö |
| Protocol: | PCR, agarose gel |
| Notes: | Primer: BBS-PstI-rv |
| Results: | no bands on the gel. Indeed, the error before occured during the pcr reaction. Thomas (supervisor) and Eni decided to give A3 assembly one more try. |
Assembling pSB1C3_Chi6 with A3 assembly
2018/08/07 - 2018/08/09| Participants: | Enikö |
| Protocol: | Restrictiondigest, Pcr purify, ligation, chem trafo, mini-prep, sequencing |
| Notes: | We switched to the enzymes EcoRI, SpeI because we realized XbaI and SpeI had the same cutting sites and might lead to religation or false direction. |
| Results: | We obtained colonies, however repeated sequencing with VF2 and VR didnt give successful reads. |
PCR to assemble pSB1C3_Chi6
2018/08/16 - 2018/08/20| Participants: | Enikö |
| Protocol: | PCR, restriction digest, pcr purify, ligation, chem trafo |
| Notes: | because oligodimerisation & ligation failed, we decided to amplify chi6 by pcr; primers: Chi6_2clone & Chi6_fill cycle: 98° 2 min; 98° 20 s; 57° 30s; 72° 20s (to 2 5 x) 72° 2 min; expect: ca 100 bp Backbone: BBS_AgeI_chi6_fw & BBP_SalI_Chi8_rv; AT: 62° expect: 2 kb next, we digested the samples with ageI, SalI |
| Results: | Didnt work, Eni ordered 3 different primer pairs to stitch chi6 together by pcr. |
preparing pSB1C3 backbone
2018/08/30| Participants: | Enikö |
| Protocol: | Restriction digest, Agarose gel, gelextraction |
| Notes: | EcoRi & SpeI |
| Results: | PIC |
dimerization of short chi primers to assemble pSB1C3_chi6
2018/08/30 - 2018/09/05| Participants: | Enikö |
| Protocol: | oligodimerization, Agarose Gel, Gel extraction, Ligation, chem trafo, PCR, mini-prep |
| Notes: | the primers were stitched together and purified from agarose gel. The samples were ligated into pSB1C3 and transformed into E.Coli NEB Turbo. The samples were tested by test-pcr via the primers VF2 and VR. |
| Results: | colonies. consequent test-pcr proved the correctness of pSB1C3_Chi6. (PIC) |