Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/02

From OpenWetWare

Jump to: navigation, search
gBlock (DBN001) & DBN002 PCR Amplification; pSB1A3-GFP Restriction Digest Main project page
Previous entry      Next entry

Overview

Running out of the gBlock ordered (DBN001), and it's not yet in a plasmid, so I'm making more by PCR amplification.

gBlock PCR run details:

Reagent Volume (uL)
2x master mix25
MB grade H2O24
gBlock f1 primer0.25
gBlock r1 primer0.25
gBlock template0.5
Total50
Step Temperature °C Duration
Initial denature95120 sec
Main Cycle (x35):
denature9530 sec
anneal6030 sec
extension7230 sec
final extension72300 sec
soak4indef.

Run is saved as DBN20140402B on the Bio-Rad thermal cycler.

DBN002 PCR Run

DBN002 f2 primer
Reagent Volume (uL) for 9 rxns (uL)
2x GoTaq MM1090
MB grade H2O9.686.4
DBN002 f2 primer0.10.9
DBN002 r1 primer0.10.9
DBN002 template0.21.8
Total20180
DBN002 f3 primer
Reagent Volume (uL) for 9 rxns (uL)
2x GoTaq MM1090
MB grade H2O9.686.4
DBN002 f3 primer0.10.9
DBN002 r1 primer0.10.9
DBN002 template0.21.8
Total20180
Step Temperature °C Duration
Initial denature95120 sec
Main Cycle (x30):
denature9530 sec
anneal49-->5630 sec
extension7290 sec
final extension72300 sec
soak4indef.

Run is saved as DBN20140402A on the Bio-Rad thermal cycler.

pSB1A3-GFP plasmid digestion

Product pSB1A3-GFP
DNA600ng (3µL)
10x FastDigest Buffer3
EcoRI1
SpeI1
Water22
Total30

Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.


Gel Electrophoresis of DBN001, DBN002 and pSB1A3-GFP Products

Gel layout:

1kb+ ladder DBN002 f2 gBlock
49C50C51C52C53C54C55C56C60C

1kb+ ladder DBN002 f3 pSB1A3-GFP
49C50C51C52C53C54C55C56Cn/a

It looks like I ran the gel for too long, so the expected size fragment for the gBlock would have run into the pSB1A3-GFP lane. Will need to run that again. It also looks as though the pSB1A3-GFP digestion was not successful. Will need to re-try with more plasmid, as I used most of it on transfecting the cells.


Personal tools