Team:Nevada/Week 5
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[[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]] | |
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<center><font size=4><b>Week 5: June 18 - June 22</font size></b></center> | <center><font size=4><b>Week 5: June 18 - June 22</font size></b></center> | ||
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+ | |||
+ | ==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 | ||
+ | |||
+ | :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) |
Latest revision as of 01:25, 27 October 2012
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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)