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

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gBlock (DBN001) & DBN002 PCR Amplification; pSB1A3-GFP Restriction Digest <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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 mix 25
MB grade H2O 24
gBlock f1 primer 0.25
gBlock r1 primer 0.25
gBlock template 0.5
Total 50
Step Temperature °C Duration
Initial denature 95 120 sec
Main Cycle (x35):
denature 95 30 sec
anneal 60 30 sec
extension 72 30 sec
final extension 72 300 sec
soak 4 indef.

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 MM 10 90
MB grade H2O 9.6 86.4
DBN002 f2 primer 0.1 0.9
DBN002 r1 primer 0.1 0.9
DBN002 template 0.2 1.8
Total 20 180
DBN002 f3 primer
Reagent Volume (uL) for 9 rxns (uL)
2x GoTaq MM 10 90
MB grade H2O 9.6 86.4
DBN002 f3 primer 0.1 0.9
DBN002 r1 primer 0.1 0.9
DBN002 template 0.2 1.8
Total 20 180
Step Temperature °C Duration
Initial denature 95 120 sec
Main Cycle (x30):
denature 95 30 sec
anneal 49-->56 30 sec
extension 72 90 sec
final extension 72 300 sec
soak 4 indef.

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

pSB1A3-GFP plasmid digestion

Product pSB1A3-GFP
DNA 600ng (3µL)
10x FastDigest Buffer 3
EcoRI 1
SpeI 1
Water 22
Total 30

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
49C 50C 51C 52C 53C 54C 55C 56C 60C

1kb+ ladder DBN002 f3 pSB1A3-GFP
49C 50C 51C 52C 53C 54C 55C 56C n/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.


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