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Team:NTU-Singapore/Notebook/8 July 2008
From 2008.igem.org
- Ran PCR for T7 ptag and SupD.
- Gel extraction of E7 (clear clean bands - yay!).
- 900-1130: Lu Chao: minipreps and nanodrops for pLacI, pFe, RBS 34, GFP (3 samples for each plasmid).
- Hung:
- 1030-1130: as the Minelute PCR purification columns are temporarily out of stock, I used the QIA PCR purification kit to purify a PCR E7 (20ul), and the GFP that was digested with EcoRI/PstI and gel-extracted on last Friday (50ul). Nanodrop showed quite low concentration and purity. However, later gel run showed clear and distinct bands for E7 (2kb) and GFP (800b).
- 1200-1215: digestion of GFP with XbaI/PstI to obtain empty plasmid vector (as Darius was also synthesizing E7 with XbaI/PstI restriction sites). Incubate at 37 degress for 4 hours (until 1615).
- 1300-1330: ligation at 1:3 ratio for:
- pFe-GFP
- LacI-GFP
Note:We tried additional step for ligation: incubate insert/vector mixture at 50 degrees for 5 mins, then put on ice for 1 min, pulse spin then start adding buffer, and quick ligase.
- 1340-1400: PCR purification for ligation mixtures. Then ligation mixtures were put on ice until transformation at 1610 (carried out together with LacI-RBS34).
- 1610-1810: Lu Chao: transformation and cell cloning for LacI-RBS 34 (use 0.5 ul of the succesfully ligated product), LacI-GFP (use both 1ul, 3ul and 4ul to transform into Homemade top10 cells) and pFe-GFP (use both 1ul, 3ul and 4ul to transform into Homemade top10 cells).
- 1620-1645: QIA purification for GFP digested with XbaI/PstI at 1200. Nanodrop showed very good concentration and purity. That means we can trust this kit to purify our digested mixtures.
- Wednesday we're trying to ligate E7 with empty plasmid using different cutting sites (EcoRI/PstI and XbaI/PstI).
- Gel Story
Pre Staining is ineffective.
- Reasons-Inconsistent Cooling process, Formation of clumps of gel during cooling, non homogenous gel led to inconsistent Gel results.
Conclusion POST STAINING
- E7 PCR Story
- E7 PCR tube Gel Run-Smeary gel
- Gel Extract-50ul-Concentrate to 30ul using PCR purification kit
- Gel run..clean clear bands at 2k mark
- Gel Extract-50ul-Concentrate to 30ul using PCR purification kit
- E7 PCR tube Gel Run-Smeary gel
- E7 PCR Direct Run Ethidium Bromide Gel
- Gel run was good.
- Confirming that post Staining of gel is good
- Gel run was good.
T7ptag Story PCR T7ptag using 1:50, 1:20, direct plasmids and T7ptag PCR as templates
SupD Story PCR SupD using 1:20 Plasmid and PCR Product as templates
