Team:NTU-Singapore/Notebook/8 July 2008

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**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.
**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).
**Wednesday we're trying to ligate E7 with empty plasmid using different cutting sites (EcoRI/PstI and XbaI/PstI).
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 +
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*Gel Story
 +
Post Staining is ineffective.
 +
**Reasons-Inconsistent Cooling process, Formation of clumps of gel during cooling, non homogenous gel led to inconsistent Gel results.
 +
Conclusion
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POST STAINING
 +
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*E7 PCR Story
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**E7 PCR tube Gel Run-Smeary gel
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***Gel Extract-50ul-Concentrate to 30ul using PCR purification kit
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****Gel run..clean clear bands at 2k mark
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*E7 PCR Direct Run Ethidium Bromide Gel
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**Gel run was good.
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***Confirming that post Staining of gel is good
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T7ptag Story
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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

Revision as of 07:29, 9 July 2008

  • 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

Post 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
  • E7 PCR Direct Run Ethidium Bromide Gel
    • Gel run was good.
      • Confirming that post Staining of gel is 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

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