Haynes Lab:Notebook/Engineering PC-TFs/2014/11/05: Difference between revisions
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'''Transformation''' | '''Normal Transformation (BL01 & BL05 in V0120)''' | ||
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# Warmed selection agar plates at 37°C. | # Warmed selection agar plates at 37°C. | ||
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# Pipetted the total volume of cells + LCR reaction onto the agar; spread using sterile glass beads. | # Pipetted the total volume of cells + LCR reaction onto the agar; spread using sterile glass beads. | ||
# Incubated overnight at 37°C. | # Incubated overnight at 37°C. | ||
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'''Long Transformation''' | |||
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Using the set up ligation products from above. | |||
*DH5α-T cells were thawed in ice. | |||
*Two 1.5mL tubes were setup and labeled; LCR BL01 & Long Transformation BL01. | |||
*60μL of thawed cells were mixed and then transferred into each of these two empty tubes. | |||
*The ligation products were then transferred into their respective tubes and flicked to mix. Set back on ice. | |||
*All tubes incubated on ice for 35 minutes. | |||
*All tubes were heat shocked on the heat block at 42°C for 30 seconds and then placed back on ice for 2 minutes. | |||
*900μL SOC Medium was pipetted into each of the three tubes. | |||
*Tubes were taped into an empty plate. The plate was taped to the shaker within the incubator. | |||
*Incubated on shaker for 1 hour at 37°C and 240rpm. | |||
*Centrifuged for 1.5 minutes at 9 x g. | |||
*500μL media removed from each tube. | |||
*Resuspended cell pellet in remaining 400μL media. | |||
*300μL cells transferred onto respective plate. | |||
*Sterile glass beads used to spread cells onto plate. | |||
*Placed in the incubator for overnight growth. | |||
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Revision as of 21:08, 5 November 2014
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Summary
Gel Extraction/Purification of BL01
Gel Purification: BL01/V0120
Concentration
LCR
Normal Transformation (BL01 & BL05 in V0120)
Long Transformation
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