Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/01: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
(Autocreate 2015/04/01 Entry for Haynes_Lab:Notebook/HPK-CFP_insertion_into_Gal4EED/Luc_using_CRISPR)
 
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==
* Insert content here...


Transformation results from last week's digestion and ligation:
* negative control: no colonies.
* positive control: numerous colonies, but non-fluorescent under UV light.
* sample: no colonies.
Could be a wrong choice of plasmid, or something went wrong during the digestion/ligation. Will repeat the process after doing a test digest on the plasmid to make sure the cut sites are there. Should see about using replacement plasmid, maybe one with RFP instead of GFP, as that's easier to visualize.
-----
DBN002 f2 and f3 primers have arrived, as well as gBlock for and rev primers. To do tomorrow:
# PCR on gBlock using primers. Run a gel to check size when done, check concentration on Take3 plate.
# PCR on DBN002 fragment from gel extraction. Make 2 products: DBN002 f2 and DBN002 f3. Use DBN002 r1 for both. Temperature gradient on both. Check annealing temp. of primers before running. Run a gel to check size of products when done.
# Restriction digest on EsaI/GFP plasmid from last week's digest/ligation. Use EcoRI and SpeI. Run a gel to check size of products when done, compare to René's notes on expected sizes. Or find it on Benchling somewhere.


<!-- ##### 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 16:31, 1 April 2015

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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Entry title

Transformation results from last week's digestion and ligation:

  • negative control: no colonies.
  • positive control: numerous colonies, but non-fluorescent under UV light.
  • sample: no colonies.

Could be a wrong choice of plasmid, or something went wrong during the digestion/ligation. Will repeat the process after doing a test digest on the plasmid to make sure the cut sites are there. Should see about using replacement plasmid, maybe one with RFP instead of GFP, as that's easier to visualize.

DBN002 f2 and f3 primers have arrived, as well as gBlock for and rev primers. To do tomorrow:

  1. PCR on gBlock using primers. Run a gel to check size when done, check concentration on Take3 plate.
  2. PCR on DBN002 fragment from gel extraction. Make 2 products: DBN002 f2 and DBN002 f3. Use DBN002 r1 for both. Temperature gradient on both. Check annealing temp. of primers before running. Run a gel to check size of products when done.
  3. Restriction digest on EsaI/GFP plasmid from last week's digest/ligation. Use EcoRI and SpeI. Run a gel to check size of products when done, compare to René's notes on expected sizes. Or find it on Benchling somewhere.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/HPK-CFP_insertion_into_Gal4EED/Luc_using_CRISPR/2015/04/01&oldid=876759"