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Team:Nevada/Week 17
From 2012.igem.org
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==September 10== | ==September 10== | ||
| + | :Michelle: | ||
| + | :::Single colony isolation of M2-BL21 from LB AMP-CM stock plate, which was followed by culturing it in 10ml LB :::AMP-CM broth and then large scale culturing with 50ml of LB AMP-CM broth. | ||
| + | |||
| + | :Justin and Dafne: | ||
| + | :::Proteins were separated from cells (as done before) | ||
| + | :::Suspended in Amylose column buffer | ||
| + | :::proteins were purified using Amylose Resin High Flow (NEB) | ||
| + | :::eluted using 1M glycine: pH 10 buffer | ||
==September 11== | ==September 11== | ||
| + | :Joe: | ||
| + | :::harvest protein from RFP-SBP | ||
| - | + | :Justin and Dafne | |
| + | :::Concentrate protein using Desalting column | ||
| + | :Jeremiah & Chris: | ||
| + | :::Express BL21 cells by use of a L. Arabanose gradient | ||
| + | :::Took and 8 hour and overnight time sample | ||
| + | |||
| + | ==September 12== | ||
| + | · Run samples on a polyacrylamide gel | ||
| + | · Western blot protocol | ||
==September 13== | ==September 13== | ||
| + | Joe: Add rice to purified protein, incubate for 5 hours | ||
| + | Not enough protein - did not work | ||
| + | Michelle: | ||
| + | |||
| + | Glycerol stocked M2-BL21 before large scale culturing M2-BL21 with 50ml LB AMP-CM broth. | ||
==September 14== | ==September 14== | ||
| + | Joe: make 10, 10 ml cultures of RFP-SBP in Top10 cells | ||
| + | |||
| + | Michelle: | ||
| + | |||
| + | Transformed M2 (SBP-LRP) into BL21 cells and plated on LB AMP-CM plates. | ||
| + | |||
| + | Jeremiah & Chris: | ||
| + | · Develop western | ||
| + | o No band appeared because the BL21 cells needed to be grown in Ampicillin (Amp) and Chloramphenicol (Cm) antibiotics | ||
| + | · Minipre fresh expression plasmid with TBP+ from BL21 culture | ||
| + | o Ran on a gel (gel #193) | ||
| + | · Transform plasmid into new BL21 cells | ||
| + | o Use Amp and Cm antibiotics when plating | ||
| + | · Prepare submission plasmid (pBP31C) | ||
| + | o Miniprep and digest in large quantities as to have enough for the other groups | ||
| + | |||
| + | |||
| + | Chris and Jeremiah: | ||
| + | |||
| + | · Colony PCR check 5 samples | ||
| + | o Ran on a gel (gel #195) | ||
Revision as of 03:54, 4 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
Contents |
September 10
- Michelle:
- Single colony isolation of M2-BL21 from LB AMP-CM stock plate, which was followed by culturing it in 10ml LB :::AMP-CM broth and then large scale culturing with 50ml of LB AMP-CM broth.
- Justin and Dafne:
- Proteins were separated from cells (as done before)
- Suspended in Amylose column buffer
- proteins were purified using Amylose Resin High Flow (NEB)
- eluted using 1M glycine: pH 10 buffer
September 11
- Joe:
- harvest protein from RFP-SBP
- Justin and Dafne
- Concentrate protein using Desalting column
- Jeremiah & Chris:
- Express BL21 cells by use of a L. Arabanose gradient
- Took and 8 hour and overnight time sample
September 12
· Run samples on a polyacrylamide gel · Western blot protocol
September 13
Joe: Add rice to purified protein, incubate for 5 hours
Not enough protein - did not work
Michelle:
Glycerol stocked M2-BL21 before large scale culturing M2-BL21 with 50ml LB AMP-CM broth.
September 14
Joe: make 10, 10 ml cultures of RFP-SBP in Top10 cells
Michelle:
Transformed M2 (SBP-LRP) into BL21 cells and plated on LB AMP-CM plates.
Jeremiah & Chris: · Develop western o No band appeared because the BL21 cells needed to be grown in Ampicillin (Amp) and Chloramphenicol (Cm) antibiotics · Minipre fresh expression plasmid with TBP+ from BL21 culture o Ran on a gel (gel #193) · Transform plasmid into new BL21 cells o Use Amp and Cm antibiotics when plating · Prepare submission plasmid (pBP31C) o Miniprep and digest in large quantities as to have enough for the other groups
Chris and Jeremiah:
· Colony PCR check 5 samples
o Ran on a gel (gel #195)
