IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/15

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dspB Track

 * Plate taken out at 11:15pm
 * no colonies
 * seems like the ligation had failed; therefore, re-do ligation between his dspB and ccdB chlor backbone

Ligation

 * Protocol: "Ligation" protocol from Biobrick
 * 5uL vector: 1.5uL his
 * Tube: his + ccdB -> HC
 * Incubate at room temperature (RT) for 20 minutes

Transformation of HC

 * Protocol: SOP; used competent cell #2
 * Changes: use 5uL instead of 1uL of ligation mix
 * Plates in 37C incubator at 3:05pm

PNK Treatment of DNA Ends on primers

 * Primers: dspB fw rxn1, dspB rev rxn1, dspB fw rxn2, dspB rev rxn2


 * Tubes: dspB fw rxn1 PNK, dspB rev rxn1 PNK, dspB fw rxn2 PNK, dspB rev rxn2 PNK

PCR using Rafael's method

 * To PCR tubes, add 3uL of rev + fw to each respective tube
 * fw+rev rxn 1 -> tube 1
 * fw+rev rxn 2 -> tube 2

Tubes: rxn1, rxn2

PCR Cycles:
 * 98C @ 2min
 * Cycle 27x:
 * 98C @ 10 sec
 * 72C @ 30 sec
 * 72C @ 40 sec
 * 72C @ 10 min
 * 10C @ hold

Start: 4:08pm End: 5:10pm

Restriction Digest
Enzymes: Use XbaI and PstI (1uL each) DNA: ccdb-chlor backbone

Digest for 2 hours

Restriction Digest
Enzymes: Use EcoRI and SpeI (1uL each) DNA: prefix and dspB NH

Digest for 2 hours

Gel verification on RD, ccdB Chlor, PCR products
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 60min Results:
 * Protocol: biobrick "digest" protocol
 * Changes: load 20uL into each well; 10uL for ladder

Gel extraction
ccdB XP gel weight: 0.1161g pre gel weight: 0.1218g
 * Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit)

Resuspended in 40uL of buffer Nanodrop:
 * [ccdB XP] = 4.6ng/uL
 * [pre] = 7.0ng/uL

PCR using Rafael's method Attempt #2

 * To PCR tubes, add 3uL of rev + fw to each respective tube
 * fw+rev rxn 1 -> tube 1
 * fw+rev rxn 2 -> tube 2

Tubes: rxn1, rxn2

PCR Cycles:
 * 98C @ 2min
 * Cycle 27x:
 * 98C @ 10 sec
 * 72C @ 30 sec
 * 72C @ 40 sec
 * 72C @ 10 min
 * 10C @ hold

Start: 9:55pm End: 10:57pm

Gel verification PCR products Attempt #2
Gel orientation: Machine conditions: 0.5x TBE buffer, 100V, 45min Results:
 * Protocol: biobrick "digest" protocol


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