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Team:Nevada/Week 14
From 2012.igem.org
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==August 23== | ==August 23== | ||
| + | :Joe: | ||
| + | :::miniprep of SBP-RFP cultures | ||
| + | :::PCR - gel 173 SBP-RFP-Tet[f] bad, Tet[f] backbone good | ||
| + | :::culture more RFP plasmid | ||
| + | |||
| + | :Michelle: | ||
| + | :::Glycerol stock 1 ml of M2-BL21 + LB AMP-CM. | ||
| + | :::Protein purification: Pellet 40 ml of M2-BL21 + LB AMP-CM, sonic pulse, follow procedure | ||
| + | :::for Nickel (Ni) column to purify protein. | ||
| + | |||
| + | :Jeremiah & Chris: | ||
| + | :::Trans formation failed due to use of the wrong cell line (BL21 cells instead of TOP 10) | ||
| + | :::Attempt transformation again | ||
==August 24== | ==August 24== | ||
Revision as of 03:40, 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 | Week 19 | Week 20 | Week 21
Contents |
August 20
- Joe:
- gel 167 - RFP pcr good, gel purify
- Digestions: SBP(SpeI/PstI), RFP(XbaI/PstI)
- Ligation of two digestions
- Michelle:
- Western blot transfer continued from 8/17 for samples of small colony from transformation of M2-BL21 developed :::membrane.
- Justin and Dafne:
- Large scale expression (200ml) of L-arabinose induced SBP-B12 protein with 5 nM vitamin B-complex
- Jeremiah & Chris:
- Western blot protocol (BL21 cells with no thiamine added)
- Run time points on a polyacrylamide gel (BL21 cells with thiamine added)
August 21
- Michelle:
- Western blot transfer of remaining supernatant from last Western blot transfer of small colony from transformation :::of M2-BL21.
- Justin and Dafne:
- Obtain protein from cells as done before
- Purify protein using Ni-NTX nickel column (per protocol)
- Analyze using western blot and coomassie brilliant blue
- Jeremiah & Chris:
- Western blot development (BL21 cells with no thiamine added)
- No band appeared after development so IPTG promoter could be the problem (picture on iphone)
- Cancel other expression attempt using IPTG and added thiamine
- Attempt to express our gene by use of a TET promoter
- Set up phusion PCR for the TBP+ gene
August 22
- Joe:
- Colony PCR - gel 171 right side both colonies good
- Michelle:
- Western blot transfer continued from yesterday for remaining supernatant sample for small colony from transformation :::of M2-BL21. Membrane developed.
- Cultured more M2-BL21 with LB AMP-CM.
- Jeremiah & Chris:
- Ran gel of phusion PCR products to confirm existence (gel #169)
- Purify gene using glass milk protocol
- Ran gel to see concentration (gel #170)
- Ligate phusion products together
- Expression plasmid phusion product already don
August 23
- Joe:
- miniprep of SBP-RFP cultures
- PCR - gel 173 SBP-RFP-Tet[f] bad, Tet[f] backbone good
- culture more RFP plasmid
- Michelle:
- Glycerol stock 1 ml of M2-BL21 + LB AMP-CM.
- Protein purification: Pellet 40 ml of M2-BL21 + LB AMP-CM, sonic pulse, follow procedure
- for Nickel (Ni) column to purify protein.
- Jeremiah & Chris:
- Trans formation failed due to use of the wrong cell line (BL21 cells instead of TOP 10)
- Attempt transformation again
