Haynes Lab:Notebook/CRISPR Editing/2014/07/09: Difference between revisions
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Rene M Davis (talk | contribs) (Autocreate 2014/07/09 Entry for Haynes_Lab:Notebook/CRISPR_Editing) |
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==07//2014== | ==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.<br><br> | |||
Will run through the protocol with pX330 and three gRNAs, g5, g13, g14<br> <br> | |||
Annealing the oligos | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''oligo 1 (100μM)''' | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
|- | |||
| oligo 2 (100μM)||1 | |||
|- | |||
| 10X T4 Ligation Buffer (NEB) ||1 | |||
|- | |||
| ddH2O||6.5 | |||
|- | |||
| T4 PNK (NEB)||0.5 | |||
|} | |||
<br> | |||
*37°C 30 min | |||
*95°C 5 min | |||
*ramp down to 25°C at 5°C/min | |||
<br><br> | |||
Ligation reaction<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''pX330 (185ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''0.540540541''' | |||
|- | |||
| 1:250 annealed oligos||2 | |||
|- | |||
| 10X Fast Digest buffer||2 | |||
|- | |||
| 10mM DTT||1 | |||
|- | |||
| 10mM ATP||1 | |||
|- | |||
| BbsI||1 | |||
|- | |||
| T7 DNA ligase||0.5 | |||
|- | |||
| water||11.95945946 | |||
|} | |||
<br> | |||
Thermocycler<br> | |||
*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 | |||
<br><br> | |||
Long transformation, 75ul cells | |||
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Revision as of 17:03, 9 July 2014
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07/09/2014Had about the same number of colonies on each of the ligation plates as the backbone. Will try the ligation following the Zhang lab protocol.
Ligation reaction
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