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

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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:

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)250
Insert DNA (211 ng/µL)015
FD Buf 10x GRN30
FD Buf 10x CLR03
XbaI11
PstI11
H2O010
Total3030

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)0.50
Vector DNA (50 ng)2.52.5
2x Roche Rapid Ligation Buffer55
NEB T4 Ligase11
H2O11.5
Total1010

Incubate at RT for 10 minutes.

Transformation

Follow the traditional transformation protocol found here.



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