Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/07
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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.
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.
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.
Going to use an excess of insert relative to vector.
Incubate at RT for 10 minutes.
Follow the traditional transformation protocol found here.