Team:Nevada/Week 10

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Week 10: July 23 - July 27

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

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