Boston University/Custom Cloning Protocol


Cloning a chromosomal gene into a plasmid

Whole-Cell PCR (3 hours) -prepare agarose gel Gel electrophoresis with SYBR safe (1 hour) -prepare Qiagen PCR Cleanup Kit Qiagen PCR Cleanup (0.5 hour) -prepare digestion plasmid Prepare digestion of PCR products (0.5 hour) Digestion of PCR products and plasmid (3 hours) -prepare Qiagen PCR Cleanup Kit -prepare agarose gel (SYBR safe) Qiagen PCR Cleanup of PCR products and run gel electrophoresis of plasmid (1 hour) -prepare Qiagen Gel Extraction Kit Qiagen Gel Extraction of plasmid from gel (0.5 hour) -prepare ligation Ligation (1 hour) -prepare competent cells from O/N culture Transformation (0.5 hour) -warm plates Plate cells

Whole-Cell PCR:

1) Pellet 75µl of O/N E.coli K12 culture in an eppy tube 2) Carefully aspirate all supernatant media 3) Put the eppy tube with the pelleted cell on ice 4) Mix the 25 µl PCR cocktail:

Control 1 Control 2 PCR Water 10.5 10.5 8.5 Primer 1 2 - 2 Primer 2 - 2 2 Master Mix 12.5 12.5 12.5 25 µl 25 µl 25 µl 5)Thermocycler 94ºC 5 min 94ºC 30 sec <------ 55ºC 1 min | repeat 35 X 65ºC 4 min <------ 65ºC 10 min 6) Prepare Gel electrophoresis analysis and PCR product cleanup (see next page)

Note that these two steps should be done at the same time by different people 1) Run 5µl of PCR product in a 1.5% agarose gel 50 ml TAE 750 mg agarose 0.5 µl 1% EtBr

120V for 45 minutes

2) Clean up rest of PCR product with a Qiagen PCR cleanup kit

PCR Purification

3) Elute with 35 µl EB instead of 50 µl 4) Measure concentration with the Nanodrop

Plasmid miniprep:

1) Miniprep using a Qiagen Miniprep Kit:

DNA Purification

DNA Purification