Team:NTU-Singapore/Notebook/1 July 2008

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Tuesday 1 July

  • Hung, Darius, Choon Kit: try out ligation of pFe(insert) with GFP(vector); and pLacI(insert) with RBS34(vector). 300 ng is used for each DNA (the volume is calculated based on concentration measured by Nanodrop on Monday).
    • 1030-1130:prepare digestion mixtures for pFe,GFP,pLacI,RBS34. Use double digestion for the inserts and sequential digestion for the vectors.
      • Vectors (1st digestion):incubate from 1130-230pm at 37 degrees C
GFP RBS34
DNA 12.6 13.7
Buffer2 2 2
BSA 0.2 0.2
Mq water 4.2 2.2
XbaI 2 2
Total 21 20
      • Inserts (double digestion):incubate from 1130-130pm at 37 degrees C
pLacI pFe
DNA 27 16.5
EcoRI Buffer 2 2
BSA 0.2 0.2
Mq water 0 10
EcoRI 1 1
SpeI 1 1
Total 31 30
    • 130pm-2pm: PCR purification (using PCR purification MinElute Kit) for pLacI,pFe to remove the enzymes (prevent star activity). After that, store the samples at 4 degrees fridge.
    • 2pm-230pm: PCR purification (using PCR purification MinElute Kit) for GFP,RBS34 to remove the enzymes (prevent star activity)
    • 230pm-6pm: 2nd digestion for GFP,RBS34 (including incubation for 3 hours). After this, we stored the samples at 4 degrees fridge.
GFP RBS34
DNA 10 (after Minelute) 10 (after Minelute)
EcoRI Buffer 2 2
Mq water 6 6
EcoRI 2 2
Total 20 20
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