Haynes Lab:Notebook/CRISPR Editing/2014/07/09

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(Autocreate 2014/07/09 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
Current revision (20:03, 9 July 2014) (view source)
(07//2014)
 
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==07//2014==
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==07/09/2014==
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Had about the same number of colonies on each of the ligation plates as the backbone. Will try the ligation following the Zhang lab protocol.<br><br>
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Will run through the protocol with pX330 and three gRNAs, g5, g13, g14<br> <br>
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Annealing the oligos
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''oligo 1 (100μM)'''
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| align="center" style="background:#f0f0f0;"|'''1'''
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|-
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| oligo 2 (100μM)||1
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|-
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| 10X T4 Ligation Buffer (NEB) ||1
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|-
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| ddH2O||6.5
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|-
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| T4 PNK (NEB)||0.5
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|}
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<br>
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*37°C 30 min
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*95°C 5 min
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*ramp down to 25°C at 5°C/min
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<br><br>
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Ligation reaction<br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''pX330 (185ng/ul)'''
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| align="center" style="background:#f0f0f0;"|'''0.540540541'''
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|-
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| 1:250 annealed oligos||2
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|-
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| 10X Fast Digest buffer||2
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|-
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| 10mM DTT||1
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|-
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| 10mM ATP||1
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|-
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| BbsI||1
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|-
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| T7 DNA ligase||0.5
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|-
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| water||11.95945946
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|}
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<br>
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Thermocycler<br>
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*37°C 5 min
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*23°C 5 min
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**Cycle the previous two steps for 6 cycles (total run time 1h)
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*4°C hold until ready to proceed
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<br><br>
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Long transformation, 75ul cells
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07/09/2014

Had about the same number of colonies on each of the ligation plates as the backbone. Will try the ligation following the Zhang lab protocol.

Will run through the protocol with pX330 and three gRNAs, g5, g13, g14

Annealing the oligos

oligo 1 (100μM) 1
oligo 2 (100μM)1
10X T4 Ligation Buffer (NEB) 1
ddH2O6.5
T4 PNK (NEB)0.5


  • 37°C 30 min
  • 95°C 5 min
  • ramp down to 25°C at 5°C/min



Ligation reaction

pX330 (185ng/ul) 0.540540541
1:250 annealed oligos2
10X Fast Digest buffer2
10mM DTT1
10mM ATP1
BbsI1
T7 DNA ligase0.5
water11.95945946


Thermocycler

  • 37°C 5 min
  • 23°C 5 min
    • Cycle the previous two steps for 6 cycles (total run time 1h)
  • 4°C hold until ready to proceed



Long transformation, 75ul cells


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