Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/07

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 cloning work and troubleshooting.

Transformation Results of KAH184 and EM7:ZeoR ligation in v0120

Negative control plate and sample plate both contain thousands of colonies. Sequencing of EM7:ZeoR_v0120 vector shows absence of EcoRI cut site; therefore the vector was only cut with XbaI and re-ligated after transformation.

Need to re-do the EM7:ZeoR cloning. Ordered new primers.

HA2_BB PCR Results

Yesterday I performed PCR to add BioBricks standard cut sites to the HA2 part. Gel image:

File:2016-01-07 gel.tif

See last two lanes. Expected size is 133bp. PCR looks like it was a success. Performed PCR clean-up and measured concentration of 211 ng/µL.

Restriction Digest

Digesting v0120 and HA2_BB separately with XbaI and PstI restriction enzymes. HA2_BB is ready to use following heat deactivation of enzymes; v0120 needs to be gel purified and quantified first.

Reagent Vector rxn (µL) Insert rxn (µL)
Vector DNA (106 ng/µL) 25 0
Insert DNA (211 ng/µL) 0 15
FD Buf 10x GRN 3 0
FD Buf 10x CLR 0 3
XbaI 1 1
PstI 1 1
H2O 0 10
Total 30 30

Incubate at 37 °C for 10 minutes. Run the vector reaction on a gel at 100 V for 1 hr, then gel purify the large fragment and quantify. Heat inactivate the insert reaction at 80 °C for 20 min.


DNA Quantification of v0120 (X&P digested)

Concentration measured to be 19.7 ng/µL. 260/280 looks good.

Ligation

Going to use an excess of insert relative to vector.

Reagent Treatment (µL) Neg. control (µL)
Insert DNA (50 ng) 0.5 0
Vector DNA (50 ng) 2.5 2.5
2x Roche Rapid Ligation Buffer 5 5
NEB T4 Ligase 1 1
H2O 1 1.5
Total 10 10

Incubate at RT for 10 minutes.

Transformation

Follow the traditional transformation protocol found here.