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

Rafael's Method

PNK Treatment of DNA Ends on primers

  • Primers: dspB fw rxn1, dspB rev rxn1, dspB fw rxn2, dspB rev rxn2
REAGENTS1 RXN VOLUME (uL)
PNK Stock1
T4 ligase buffer1
Primer8
  • Tubes: dspB fw rxn1 PNK, dspB rev rxn1 PNK, dspB fw rxn2 PNK, dspB rev rxn2 PNK

PCR using Rafael's method

PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP1
sdH2O6.65
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
gDNA3
Total25
  • 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
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)
Buffer 22
BSA0.2
DNA10
ddH205.8
Total20

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

Digest for 2 hours

Assembling the final construct

Restriction Digest
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL)
Buffer 22
BSA0.2
DNA10
ddH205.8
Total20

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

Digest for 2 hours

Gel verification on RD, ccdB Chlor, PCR products

  • Protocol: biobrick "digest" protocol
  • Changes: load 20uL into each well; 10uL for ladder

Gel orientation:

Gel orientation
1 kb ladderpredspB100bp ladder12ccdB XP

Machine conditions: 0.5x TBE buffer, 100V, 60min Results:

Gel extraction

  • Using Invitrogen PureLink Quick Gel Extraction Kit (protocol inside kit)

ccdB XP gel weight: 0.1161g
pre gel weight: 0.1218g

Resuspended in 40uL of buffer Nanodrop:

  • [ccdB XP] = 4.6ng/uL
  • [pre] = 7.0ng/uL

PCR using Rafael's method Attempt #2

PCR Master mix
Reagent1x rxn volume (uL)
5x rxn buffer5
10mM dNTP1
sdH2O6.65
Phusion polymerase0.1
MgCl22
DMSO - 5%1.25
gDNA3
Total25
  • 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

  • Protocol: biobrick "digest" protocol

Gel orientation:

Gel orientation
Quick load 100 bp ladder(last year)2bWQuick load 100bp ladder (this year)12ccdB XP

Machine conditions: 0.5x TBE buffer, 100V, 45min Results:


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