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Team:Nevada/Week 10
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 |
July 23
- Joe:
- PCR of RFP gene
- Digestions: SBP by SpeI and PstI, RFP by XbaI and PstI
- Ligation of the two digests
- Michelle:
- Check Western blot transfer and sequencing from Nevada Genomic Sequencing Center of
- colony #2 from transformation of SBP (SpeI and PstI)-LRP (PstI and XbaI) digested with
- XbaI and PstI ligated with EP.
- Miniprep more SBP-LRP plasmid.
- Jeremiah & Chris:
- Digest TBP
- XbaI and PstI
- Digest TBP+++
- SpeI and XbaI
- Digest TBP
July 24
- Joe:
- Transformation of SBP-RFP, plate on Kana. plates
- Michelle:
- Run gel of 7/13 SBP (SpeI and PstI)-LRP (PstI and XbaI) digested with XbaI and Pst I.
- PCR SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid with sense primer (DNA2PTF sense)
- 10x) and antisense primer (DNA2PTF anti) (10x).
- Cultured 2 colonies of CP-EP* L-arabinose with TB-AMP.
- Justin and Dafne:
- Use pellets acquired from expression
- Suspend in lysis buffer
- Add 500 ul triton 100x
- Pellet cells
- Repeat 3x
- After final spin-down, suspend pellet in 1.5 ml of 8M urea buffer
- Allowed to incubate for 2 hours
- Spin-down, transfer supernatant to clean tube
- Analyzed supernatant with Western Blot
July 25
- Joe:
- Colony PCR all 5 white colonies of SBP-RFP cells
- Culture colonies 1 and 5
- Michelle:
- PCR purification of PCR SBP (SpeI and PstI)-LRP (PstI and XbaI) plasmid with sense primer
- DNA2PTF sense) (10x) and antisense primer (DNA2PTF anti) (10x) from yesterday and
- Digest with XbaI and PstI.
- Miniprep culture of CP-EP* L-arabinose for stock.
July 27
- Jeremiah & Chris:
- Ligate SBP-TBP into ExV (L. Arabinose)
- Justin and Dafne:
- Analyzed Western Blot
- Band revealed that the protein in the inclusion body is soluble in 8M urea
July 26
- Joe:
- Analyze TET/IPTG sequences
- Retrieve promoters from cartridge: TET and IPTG
- Transform both promoters, plate on AMP
- Dafne and Michelle:
- PCR colony SBP (SpeI and PstI)-LRP (PstI and XbaI) with XbaI and PstI digest from
- yesterday before ligating to L-arabinose induced EP and transformation onto AMP plates.
- Jeremiah & Chris:
- Digest SBP-TBP and ExV and ligate again.
July 27
- Joe:
- Culture TET* and IPTG* colonies in AMP
- SBP-RFP- miniprep, digest by XbaI/PstI as well as by SpeI/XbaI
- Ligation of SBP-RFP(XbaI/PstI) with L-arabinose plasmid
- Jeremiah & Chris:
- Transform ligation from 07/26
- Justin and Dafne
- Due to urea’s strong denaturing characteristics, SBP-B12 protein needed to be refolded
- Dialysis was utilized to gradually decrease the pH of the SBP-B12 solution
- Placed in Dialysis membrane surrounded by 8M urea buffer
- this was allowed to sit overnight
