Difference between revisions of "Team:Munich/chibiobrick2.html"
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<td>Participants:</td> | <td>Participants:</td> | ||
| − | <td>Enikö</td> | + | <td>Enikö Baligács</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Protocol:</td> | <td>Protocol:</td> | ||
| − | <td> | + | <td> |
| + | <a href="https://static.igem.org/mediawiki/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restrition digest</a>, | ||
| + | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
| + | <a href="https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020" target="_blank">Gel extraction</a> | ||
| + | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| Line 195: | Line 199: | ||
<td>Participants:</td> | <td>Participants:</td> | ||
| − | <td>Enikö</td> | + | <td>Enikö Baligács</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Protocol:</td> | <td>Protocol:</td> | ||
| − | <td> | + | <td> |
| + | <a href="https://www.sigmaaldrich.com/technical-documents/protocols/biology/annealing-oligos.html" target="_blank">Oligodimerization</a>, | ||
| + | <a href="https://static.igem.org/mediawiki/2018/b/be/T--Munich--WL1_Agarose-Gel_electrophoresis.pdf" target="_blank">Agarose gel</a>, | ||
| + | <a href="https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020" target="_blank">Gel extraction</a>, | ||
| + | <a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>, | ||
| + | <a href="https://static.igem.org/mediawiki/2018/a/a5/T--Munich--WL1_Chemical_transformation.pdf" target="_blank">Chemical Transformation</a>, | ||
| + | <a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>, | ||
| + | <a href="https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" target="_blank">Mini Prep</a> | ||
| + | </td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
Revision as of 16:57, 15 October 2018
Forming oligodimers from single-strand DNA chi6 sequences
2018/07/31| Participants: | Dominic Schwarz |
| Protocol: | Oligodimerization |
Assembling pSB1C3_Chi6 with A3 Assembly
2018/08/01| Participants: | Dominic Schwarz |
| Protocol: | Restrition digest, PCR purification, Gibson assembly, Chemical Transformation |
| Notes: | Restriction digest with XbaI, SpeI-HF |
| Results: | no colonies |
Redo: Assembling pSB1C3_Chi6 with Ligation
2018/08/02 - 2018/08/06| Participants: | Enikö Baliács |
| Protocol: | PCR, PCR purification, Gibson assembly, Ligation, Chemical transformation, 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 Baligács |
| Protocol: | PCR, Agarose gel 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 Baligács |
| Protocol: | Restrition digest, PCR purification, Ligation, Chemical transformation, Mini Prep, Sequncing |
| 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 Baligács |
| Protocol: | PCR, Restrition digest, PCR purification, Ligation, Chemical transformation |
| 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 Baligács |
| Protocol: | Restrition digest, Agarose gel, Gel extraction |
| Notes: | EcoRi & SpeI |
| Results: | PIC |
dimerization of short chi primers to assemble pSB1C3_chi6
2018/08/30 - 2018/09/05| Participants: | Enikö Baligács |
| Protocol: | Oligodimerization, Agarose gel, Gel extraction, Ligation, Chemical Transformation, 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) |