Haynes Lab:Notebook/CRISPR Editing/2016/02/29: Difference between revisions
Rene M Davis (talk | contribs) |
Rene M Davis (talk | contribs) |
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==02/29/2015== | ==02/29/2015== | ||
Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow<br><br> | Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow<br><br> | ||
'''Ligation for pX330_dCas9_EGFP'''<br> | |||
Backbone calculations for 50ng | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''conc (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''length (bp''' | |||
| align="center" style="background:#f0f0f0;"|'''ul for 50ng''' | |||
|- | |||
| pX330A_dCas9_1x4||52||8939||0.96 | |||
|} | |||
insert calculations for 1:2 ratio | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''conc (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''length (bp''' | |||
| align="center" style="background:#f0f0f0;"|'''ul for 2:! ratio''' | |||
|- | |||
| gB009||1.60||838.00||5.86 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''ul''' | |||
|- | |||
| backbone||0.96 | |||
|- | |||
| insert or water||5.86 | |||
|- | |||
| NEB 10x ligation buffer||2 | |||
|- | |||
| NEB T4 ligase||1 | |||
|- | |||
| water||10.18 | |||
|- | |||
| total||20 | |||
|} | |||
Mixed gently, inc at RT for 30 mins<br>Add 50ul cells to each reaction<br>inc on ice for 30min, heat shock at 42 for 35s, ice for 2 mins, shake in 750ul SOC for 1 hour, plate | |||
<br><br> | |||
'''Ligation for MV14'''<br> | |||
Backbone | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''conc (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''length (bp''' | |||
| align="center" style="background:#f0f0f0;"|'''ul for 50ng''' | |||
|- | |||
| MV13-KAH60||48||6240||1.04 | |||
|} | |||
Insert | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''conc (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''length (bp''' | |||
| align="center" style="background:#f0f0f0;"|'''ul for 2:! ratio''' | |||
| align="center" style="background:#f0f0f0;"|'''ul 1:100 dilution''' | |||
|- | |||
| gB014||64||122||0.03||3.05 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''ul''' | |||
|- | |||
| backbone||1.04 | |||
|- | |||
| insert or water||3.05 | |||
|- | |||
| NEB 10x ligation buffer||2 | |||
|- | |||
| NEB T4 ligase||1 | |||
|- | |||
| water||12.9 | |||
|- | |||
| total||20 | |||
|} | |||
Mixed gently, inc at RT for 30 mins<br>Add 50ul cells to each reaction<br>inc on ice for 30min, heat shock at 42 for 35s, ice for 2 mins, shake in 750ul SOC for 1 hour, plate<br><br> | |||
'''Running gel for western'''<br> | '''Running gel for western'''<br> |
Revision as of 14:43, 29 February 2016
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
02/29/2015Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow
insert calculations for 1:2 ratio
Mixed gently, inc at RT for 30 mins
Ligation for MV14
Insert
Mixed gently, inc at RT for 30 mins Running gel for western Gel lanes
Mastermix for sample buffer
heat at 100degC for 5 mins, cool in rack at RT
remove the strip from the gel, put premade gel into box with the open part facing the outer chamber, fill inner chamber to cover the comb, outer chamber to cover the bottom of the gel.
run gel for 1 hour at 120V (until lowest dye band ran just past the third ridge in gel box)
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