Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/21
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Cloning of DBN001 (gBlock insert) into mCh_pSB1A3Trying the ligation again (previous attempt), with major revisions as suggested by Dr. Haynes. Remember to use one negative control (water), one negative control (digested backbone), and one positive control (untreated vector). Major steps:
mCherry vector concentration is 392 ng/µL. gBlock concentration is 311 ng/µL. --Restriction Digest-- In two separate tubes, digest the DBN001 (gBlock) and the vector using the following reagents. Use plenty of DNA for this step; the objective is to have an abundance of digested material to work with downstream.
Mix and incubate at 37°C for 15 minutes. Skip heat inactivation (you'll be purifying the restriction digest products). --'PCR' Clean-up of digested DBN001-- Follow the instructions in the PCR clean-up kit. --Gel purification of mCh_pSB1A3 backbone-- Load entire volume of digest into one lane of the gel. Gel image: Mass of band fragment excised from gel: ____mg
--DNA Quantification--
--Ligation-- Ligate using Roche DNA Ligase buffer and NEB T4 Ligase.
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes. --Transformation-- Set aside 2µL of ligated plasmid for gel electrophoresis. Transform cells with entire remaining volume of ligated plasmid. Remember to use negative and positive controls (water and untreated vector, respectively).
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