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Team:Nevada/Week 18
From 2012.igem.org
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[[Team:Nevada/Week 17| Week 17]] | | [[Team:Nevada/Week 17| Week 17]] | | ||
[[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/Final Weeks| Final Weeks]] | | ||
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==September 17== | ==September 17== | ||
| + | :Joe: | ||
| + | :::purify protein from 10, 10ml tubes | ||
| + | :::incubate with rice for 5 hours | ||
==September 18== | ==September 18== | ||
| + | |||
| + | Joe: RFP-SBP ligation for submission | ||
| + | culture more RFP-SBP in Top10 cells | ||
| + | Successful sticky of protein to rice, got pictures | ||
| + | |||
| + | Michelle: | ||
| + | :::Small cultured two successful colonies from M2-BL21 transformation with LB AMP-CM. | ||
| + | |||
| + | |||
| + | :Justin and Dafne: | ||
| + | :::Express more SBP-B12 protein | ||
| + | :::Using 96-well binding plate, add 100 ul of concentrated protein to 10 wells | ||
| + | :::incubate overnight | ||
==September 19== | ==September 19== | ||
| + | :Joe: Transform RFP-SBP for submission | ||
| + | |||
| + | Justin and Dafne | ||
| + | |||
| + | Rinse plate 5x using PBS-T with 5% non-fat milk | ||
| + | Add 100 ul of blocking buffer (PBS-T with 5% non-fat milk) to 10 wells with protein as well as 10 wells with no protein (control) | ||
| + | Incubate overnight | ||
| + | Purify yesterdays protein expression using amylose column (as done before) | ||
| + | Digest SBP, B12, SBP-B12, SBP-B12 in expression plasmid by EcoRI HF and Pst I HF | ||
| + | |||
==September 20== | ==September 20== | ||
| + | Joe: Culture more RFP | ||
| + | Digest SBP by EcoRI/PstI for submission | ||
| + | RFP-SBP: make 10 tubes from glycerol stock | ||
| + | Colony PCR of RFP-SBP for submission | ||
| + | Michelle: | ||
| + | |||
| + | PCR colony check M2-BL21 | ||
| + | Forward Primer (SBP-sense124)(10x diluted) and Reverse Primer (antilysine)(10x diluted) before large scale culturing | ||
| + | brightest colony with 100ml LB AMP-CM and making glycerol stocks. | ||
| + | |||
| + | Results: | ||
| + | PCR colony was very successful showed that both colonies in lanes #2 and #3 had bright bands [Gel #198]. Therefore indicating large scale culturing could be proceeded. | ||
| + | |||
| + | Justin and Dafne | ||
| + | |||
| + | Add 10 ul of vitamin B12-HRP to 1 ml of PBS-T | ||
| + | Make 4 more 100 fold dilutions of above solution | ||
| + | Add each diluted solution to 2 wells | ||
| + | incubate overnight | ||
| + | Run Western blot analysis and Coomassie stain analysis of purified protein from yesterday | ||
| + | Ligate yesterday’s digestions into submission plasmid, transform into TOP10 cells | ||
| + | |||
| + | Jeremiah & Chris: | ||
| + | · Express the TBP+ gene using an L. Arabinose gradient | ||
| + | · Digest TBP, SBP, TBP+, TBP+++, and LA-TBP+ | ||
| + | o Run on gel (gel #200) | ||
| + | · Ligate the genes above into pSB1C3 | ||
| + | · Make glycerol stock of LA-TBP+ in BL21 cells | ||
==September 21== | ==September 21== | ||
| + | Michelle: | ||
| + | |||
| + | Digest LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM with EcoRI and PSTI | ||
| + | followed by ligation into PSBC13 (digested with EcoRI and PSTI) | ||
| + | transformation onto LB-CM plates. | ||
| + | |||
| + | Results: | ||
| + | LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM into PSBC13 for iGEM submission. | ||
| + | |||
| + | Justin and Dafne | ||
| + | |||
| + | Rinse wells with PBS-T 5x | ||
| + | Rinse well with PBS 3x | ||
| + | Add TMB-Microwell peroxidase to each well | ||
| + | Digest plasmids again | ||
| + | |||
| + | |||
| + | Jeremiah & Chris: | ||
| + | · Purify pSB1C3 again via glass milk purification | ||
| + | o Run on gel (gel #203) | ||
| + | · Dephosphorylate pSB1C3 | ||
| + | LA-TBP+ PCR check | ||
Latest revision as of 01:44, 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 |
September 17
- Joe:
- purify protein from 10, 10ml tubes
- incubate with rice for 5 hours
September 18
Joe: RFP-SBP ligation for submission
culture more RFP-SBP in Top10 cells Successful sticky of protein to rice, got pictures
Michelle:
- Small cultured two successful colonies from M2-BL21 transformation with LB AMP-CM.
- Justin and Dafne:
- Express more SBP-B12 protein
- Using 96-well binding plate, add 100 ul of concentrated protein to 10 wells
- incubate overnight
September 19
- Joe: Transform RFP-SBP for submission
Justin and Dafne
Rinse plate 5x using PBS-T with 5% non-fat milk Add 100 ul of blocking buffer (PBS-T with 5% non-fat milk) to 10 wells with protein as well as 10 wells with no protein (control) Incubate overnight Purify yesterdays protein expression using amylose column (as done before) Digest SBP, B12, SBP-B12, SBP-B12 in expression plasmid by EcoRI HF and Pst I HF
September 20
Joe: Culture more RFP
Digest SBP by EcoRI/PstI for submission RFP-SBP: make 10 tubes from glycerol stock Colony PCR of RFP-SBP for submission
Michelle:
PCR colony check M2-BL21 Forward Primer (SBP-sense124)(10x diluted) and Reverse Primer (antilysine)(10x diluted) before large scale culturing brightest colony with 100ml LB AMP-CM and making glycerol stocks.
Results: PCR colony was very successful showed that both colonies in lanes #2 and #3 had bright bands [Gel #198]. Therefore indicating large scale culturing could be proceeded.
Justin and Dafne
Add 10 ul of vitamin B12-HRP to 1 ml of PBS-T Make 4 more 100 fold dilutions of above solution Add each diluted solution to 2 wells incubate overnight Run Western blot analysis and Coomassie stain analysis of purified protein from yesterday Ligate yesterday’s digestions into submission plasmid, transform into TOP10 cells
Jeremiah & Chris: · Express the TBP+ gene using an L. Arabinose gradient · Digest TBP, SBP, TBP+, TBP+++, and LA-TBP+ o Run on gel (gel #200) · Ligate the genes above into pSB1C3 · Make glycerol stock of LA-TBP+ in BL21 cells
September 21
Michelle:
Digest LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM with EcoRI and PSTI followed by ligation into PSBC13 (digested with EcoRI and PSTI) transformation onto LB-CM plates.
Results: LRP, SBP+LRP, and TET-RBS-SBP-LRP-TERM into PSBC13 for iGEM submission.
Justin and Dafne
Rinse wells with PBS-T 5x Rinse well with PBS 3x Add TMB-Microwell peroxidase to each well Digest plasmids again
Jeremiah & Chris:
· Purify pSB1C3 again via glass milk purification
o Run on gel (gel #203)
· Dephosphorylate pSB1C3
