User:Mara Peterson/Notebook/Mara Peterson/2015/12/14
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Performing restriction digest on parts HA1 and EM7:ZeoR, as well as restriction digest on v0120 plasmid, followed by gel extraction. Then performing a ligation reaction followed by transformation of competent E. coli cells.
Homology Arm 1: amplified using P040 and P137, contains EcoRI and SpeI restriction sites. Concentration: 6 ng/µL
Following the Assembly 101 procedure, for the most part.
Digested at 37°C for 10 minutes, followed by heat inactivation at 80°C for 5 minutes.
Cut out the two large v0120 bands (backbone) and performed gel purification using the Sigma kit. Measured concentration on the nanodrop system...
Vector volume calculations:
Insert volume calculations: Insert Insert amt (ng) Insert conc. (ng/µL) Insert vol. (µL) HA1 E/S 3.7 6.1 0.61 EM7:ZeoR X/S 13.87 37.8 0.37
I'm going to err on the side of caution with the insert volumes and aliquot 2 µL for each one instead of the 0.3-0.6 µL recommended.
Incubate at room temperature, 10 minutes.