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Team:Nevada/Week 5
From 2012.igem.org
Protocols | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Final Weeks |
Contents |
June 18
- Jeremiah & Chris:
- Ran gel of colony PCR sample (001)
- Cultured both standard and Roche colonies. (later found that colonies didn’t contain desired plasmids)
- Cultured VB12 from stock plasmid in gel in LB+Amp
- Purified VB12
- Digested SBP-TBP and VB12 to ligate together again.
- SBP-TBP: SpeI & PstI
- VB12: XbaI & PstI
- Ligated VB12àSBP-TBP
- Jasmine:
- Created control transformations and plated onto Amp plates:
- Expression plasmid alone
- EP digested with SpeI
- Self-ligated EP digest
- Dephosphorylated EP digest
- Created control transformations and plated onto Amp plates:
- Michelle and Joe:
- PCR purification of RFP plasmid and run through gel.
- PCR purified RFP plasmid again because sterile water was not used.
- After PCR purification of RFP, ligation using Invitrogen TA ligation kit and transformation into competent cells onto AMP plates was carried out.
June 19
- Jasmine:
- Checked control transformations
- Michelle and Joe:
- Check transformation of RFP TA AMP plate.
- PCR colony check 9 colonies from RFP TA transformation and ran through a gel.
- Cultured colony #2 from RFP TA transformation using Terrific broth-Ampicillin (TB-AMP).
- Cultured more SBP + LRP from 6/4 colony #2 from successful transformation SBP (SpeI and PstI) and LRP (PstI and XbaI) with TB-KAN for stock.
- Transformation of 6/4 colony #2 from transformation SBP (SpeI and PstI) and LRP (PstI and XbaI) into competent cells followed by transformation onto KAN plates for stock.
- Jeremiah & Chris:
- Transformed SBP-TBP-VB12
- Digested SBP-TBP to be used in future ligations with XbaI & SpeI
June 20
- Jasmine:
- Amplified more RFP using Phusion PCR
- Michelle and Joe:
- Miniprep cultured colony #2 from RFP-TA and digested with XbaI and PstI.
- Ran RFP-TA colony #2 digested with XbaI and PstI, followed by PCR purification, PCR
- Check with gel, and ligation using Invitrogen TA ligation kit.
- Check transformation of colony #2 from SBP (SpeI and PstI) and LRP (PstI and XbaI).
- Cultured colonies from transformation of colony #2 from SBP (SpeI and PstI) and LRP (PstI and XbaI) with TB-KAN for stock.
- Jeremiah & Chris:
- Retransformed SBP-TBP-VB12 as first transformations didn’t take.
- Ligated SBP-TBP into expression vector.
June 21
- Michelle and Joe:
- Transformation of ligation of RFP-TA colony #2 (XbaI and PstI) digest from yesterday into competent cells and onto X-GAL plates.
- Pellet and miniprep SBP + LRP. Digest SBP + LRP with XbaI, EcoRI, SpeI, and PstI for expression.
- PCR purify 20ul of digest and elute with 50ul ddH20, then run through gel.
- Redigest SBP + LRP with XbaI, EcoRI, SpeI, and PstI again.
- Jeremiah & Chris:
- Digestion of SBP-TBP to try ligation again with VB12
- Ligated SBP-TBP into expression vector (TSAP)
- Justin and Dafne:
- Ligate SBP-B12 insert into expression plasmid
- Transform ligation into TOP 10 competent cells
- Results still negative
June 22
- Michelle and Joe:
- Check transformation of RFP-TA colony #2 (XbaI and PstI) followed by PCR colony check.
- Run redigested SBP + LRP (XbaI, EcoRI, SpeI, and PstI) through gel. PCR purification using 40ul of digest and elute twice with 15ul ddH20. Ligate to expression plasmid (EP).
- Jeremiah & Chris:
- Transformation of SBP-TBP à ExV
- Ligation of VB12 à SBP-TBP
- Colony PCR of transformation from 6/15 (gel 027)
