DpnI Digest
Ran DpnI restriction digest on products from PCR of TEVp (with 15aa linker), essentially following protocol from 2014 June 24.
Conditions for DpnI digest:
Chemical | 15aa-TEVp |
|---|---|
| Starting PCR reaction | 24 uL |
| DpnI | 1.7 uL |
| NEBuffer 2.1 | 3.4 uL |
| dH2O | 5.9 uL |
| TOTAL | 35 uL |
Incubated at 37C for 45 minutes. Heat shock at 80C for 20 minutes.
Transformation of Golden Gates
Transformed ultra-competent E. coli cells with results of the following Golden Gates: CD79A+stop, CD79A-TCS-Gal4VP16, Syk+stop
Used protocol from Transformation, except plated all 0.5 mL of culture on each plate. Next time, this should be spun down before plating. Also, used 37C incubators in all relevant steps.
Also transformed ultra-competent and NEB E. coli cells with pUC19 control. For each, plated two different plates, one with 200uL of culture and another with 2uL of culture (diluted into 50uL of SOC for ease of plating).
Plates placed in 37C incubator at 7pm. Plates moved to 4C refrigerator at 8:45am.
Additional Golden Gates
Golden Gates of Lyn+stop, Lyn-mKate, Lyn-TEV, Syk-TEV, Syk-eYFP
See attached Excel file for volumes used in Golden Gates performed today: golden gate 6-26.xlsx
Left in incubator overnight.