Haynes Lab:Notebook/CRISPR Editing/2014/07/04: Difference between revisions
Rene M Davis (talk | contribs) (Autocreate 2014/07/04 Entry for Haynes_Lab:Notebook/CRISPR_Editing) |
Rene M Davis (talk | contribs) |
||
| Line 7: | Line 7: | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==Entry title== | ==Entry title== | ||
Annealing gRNA oligos<br> | |||
3ul 100uM top oligo<br> | |||
3ul 100uM bottom oligo<br> | |||
2ul 10x annealing buffer<br> | |||
12ul H2O<br> | |||
20 ul reactions <br><br> | |||
Assemblies<br> | |||
boil and cool method<br> | |||
Aluminum foil lid on the beaker to prevent condensation<br> | |||
Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).<br> | |||
Float the oligo mixtures in the boiling water for 10 min. Cover the beaker with aluminum foil to keep the air above the tube warm.<br> | |||
Turn off the heat source and allow the aluminum foil-covered water bath to slowly cool to room temperature (25°C) for several hours.<br><br> | |||
PCR of gBlock of mCh donor sequence with stop codons at three spots. After PCR with three sets of primers, will have three donor sequences each with two stop codons in different locations. <br> | |||
Ran 2-100ul PCR reactions for each primer set, 51°C annealing temp, 30s extension time.<br><br> | |||
Digest of pX330 and pX335. 4ug of each in 80ul reaction, 4ul BbsI. Left at 37°C for about 25 minutes. gel extracted<br><br> | |||
also ran mCh donor sequence on the gel, didn't see any bands but it's possible I didn't have enough EtBr because all my bands were faint. The bands should be pretty small so even though they're PCR products, they still might be faint. Didn't think to soak the gel. will rerun<br><br> | |||
<br><br> | |||
Ligations | |||
<br> | |||
Calculated volume for 50ng of each backbone. Added the volume of annealed inserts that karmella added in the protocol I followed. I'm not sure if I should try to vary the volume but that would give me tons of reactions. This can be an option in the future if I have trouble ligating these.<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''pX330''' | |||
| align="center" style="background:#f0f0f0;"|'''pX335''' | |||
|- | |||
| insert||0.5||0.5 | |||
|- | |||
| backbone||1.33||1.06 | |||
|- | |||
| 2x ligase buffer||5||5 | |||
|- | |||
| ligase||1||1 | |||
|- | |||
| water||2.15||2.5 | |||
|- | |||
| Total||10||10 | |||
|} | |||
<br><br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Backbone''' | |||
| align="center" style="background:#f0f0f0;"|'''Insert''' | |||
|- | |||
| pX330||bb ctrl | |||
|- | |||
| pX330||g14 | |||
|- | |||
| pX330||g5 | |||
|- | |||
| pX330||g13 | |||
|- | |||
| pX330||gn1T | |||
|- | |||
| pX330||gn1B | |||
|- | |||
| pX330||gn3T | |||
|- | |||
| pX330||gnBb B | |||
|- | |||
| pX335||bb ctrl | |||
|- | |||
| pX335||gn3T | |||
|- | |||
| pX335||gn1B | |||
|- | |||
| pX335||gn1T | |||
|- | |||
| pX335||gn3bB | |||
|} | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | ||
Revision as of 14:12, 8 July 2014
 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> | |||||||||||||||||||||||||||||||||||||||||||||||||
Entry titleAnnealing gRNA oligos 3ul 100uM top oligo 20 ul reactions
Digest of pX330 and pX335. 4ug of each in 80ul reaction, 4ul BbsI. Left at 37°C for about 25 minutes. gel extracted
| ||||||||||||||||||||||||||||||||||||||||||||||||||
