Team:Nevada/Week 10
From 2012.igem.org
(Difference between revisions)
Leochong718 (Talk | contribs) |
|||
(2 intermediate revisions not shown) | |||
Line 26: | Line 26: | ||
[[Team:Nevada/Week 18| Week 18]] | | [[Team:Nevada/Week 18| Week 18]] | | ||
[[Team:Nevada/Week 19| Week 19]] | | [[Team:Nevada/Week 19| Week 19]] | | ||
- | [[Team:Nevada/ | + | [[Team:Nevada/Final Weeks| Final Weeks]] | |
- | + | ||
</b></div> | </b></div> | ||
<html> | <html> | ||
Line 35: | Line 34: | ||
==July 23== | ==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== | ==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== | ==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== | ==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== | ==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 |
Latest revision as of 01:36, 27 October 2012
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