Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/08

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.pngToday's project is... Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

Overview

More adventures in cloning...

To do:

  • quantify DNA of the four DBN010 plasmid preps
  • quantify DNA of HA1_v0120 plasmid (for future ligation)
  • restriction digest of DBN010 (EcoRI & XbaI)
  • gel purification of DBN010 (3700 bp fragment)
  • quantify DNA of E&X digested DBN010
  • ligation: KAH184 (E&S) with DBN010 (E&X)
  • transformation (quick) into DH5α-Turbo

DNA Quantification

quantification went fine, concentrations are between 50 and 70 ng/µL for DBN010. A bit lower for HA1_v0120, between 7 and 24 ng/µL. 260/280 values are good.

Restriction Digest

Reagent Volume (µL)
DNA (DBN010) 25
FD 10x GRN 3
EcoRI 1
XbaI 1
Total 30

Incubate at 37 °C for 10 minutes, then purify via gel electrophoresis.

Gel purification

Gel image:

File:2016-02-08 DBN010 E&X digest.tif

All four plasmids appear to contain the insert. Will proceed with gel extraction from colonies 1 and 3.

DNA Quantification, post-purification

Concentration is ~7 ng/µL for both samples. 260/280 is on the high side, around 2.8-2.9. Going to proceed with ligation...

Ligation

Reagent Volume (µL) Negative
Vector (50 ng) 7.1 7.1
Insert (2:1) 3.1 0.0
10x buffer 1.2 1.0
ligase 1.0 1.0
H2O 0.0 0.9
Total 12.4 10.0

Incubate at room temperature for 10 minutes, then take 1 µL from each sample (treatment & negative control) and use for quick transformation protocol.