Team:HKU HongKong/Data/Mol Protocols.html
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Latest revision as of 01:02, 27 September 2012
Team:HKU HK
From 2011.igem.org
Molecular Cloning Protocols
Transforming Competent E.coli with the
Plasmid:
Notes:
-
Colonies grown on plate were selected for colony PCR screening. Miniprep to Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit):
Midiprep for Large Volumes of Culture (QIAGEN Plasmid Midi Kit): Refer to the QIAGEN website for the protocol. PCR Amplification of Gene from Bacterial Genome/Standard Biobrick Parts
37.5μL ddH2O - For sequential PCR reactions: After the first PCR is complete, perform PCR clean-up. Then use the product to conduct the second PCR reaction, after which gel purification needs to be carried out. - The volume of DNA used in the second PCR reaction depends on the concentration of the DNA after the PCR Clean-up. PCR Clean-Up – Qiagen QIAquick PCR Purification:
14.52μL ddH2O Notes:
-
The
mixture was pipetted into PCR tubes
95°C - 30 sec
Gel Purification of DNA (Qiagen QIAquick Gel Extraction Kit)
Determining DNA Concentration Using NanoDrop Spectrophotometry
__μL ddH2O (to a total of 40uL) Notes:
-
The NEB official website should be checked for buffers suitable for each
restriction enzyme. Results can vary depending on double or single
digestion. Dephosphorylation of 5' Ends of Vector Backbone -
Add
0.5µL (0.5 units per 1 ug DNA) of Calf Intestinal Alkaline Phosphatase
(CIAP) to the digested sample
immediately after digestion.
__μL
Autoclaved ddH2O (to a total of 20uL) Notes:
-
Insert and Vector must be in a 3:1 ratio. The amount of each depends on
their concentration (ng/uL) and legnth (bp). PCR Deletion (Site-Directed Mutagenesis) Reaction Note: Keep everything on ice and add all volumes in a PCR tube.
?
µL ddH2O (? = whatever volume needed to bring the total volume up to
50µL) - Volumes of diluted primer based on calculations for our ng/µL concentrations
95°C for 2min |
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