Difference between revisions of "Team:Munich/chibiobrick2.html"

Revision as of 12:25, 16 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:	Restriction 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
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:	Restriction 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, Restriction 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:	Restriction 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)

@@ Line 29: / Line 29: @@
        <td>Protocol:</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/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restriction digest</a>,
 <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,
 <a href="https://international.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510" target="_blank">Gibson assembly</a>,
@@ Line 115: / Line 115: @@
        <td>Protocol:</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/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restriction digest</a>,
 <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,
 <a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,
@@ Line 145: / Line 145: @@
        <td>
 <a href="https://static.igem.org/mediawiki/2018/3/3c/T--Munich--WL1_PCR.pdf" target="_blank">PCR</a>,
-<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/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restriction digest</a>,
 <a href="https://international.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol" target="_blank">PCR purification</a>,
 <a href="https://static.igem.org/mediawiki/2018/1/1a/T--Munich--WL1_Ligation.pdf" target="_blank">Ligation</a>,
@@ Line 177: / Line 177: @@
        <td>Protocol:</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/2018/7/7e/T--Munich--WL1_Restriction_Digest.pdf" target="_blank">Restriction 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>