Boston University/Custom Cloning Protocol
From 2007.igem.org
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[[Image:BU_DNA Purification3.jpg|center|DNA Purification]] | [[Image:BU_DNA Purification3.jpg|center|DNA Purification]] | ||
| + | |||
| + | Note that PCR product and vector digestion can be run simultaneously | ||
| + | |||
| + | PCR product digestion: | ||
| + | 1) Prepare 20 µl digestion reaction: | ||
| + | 2 µl EcoRI buffer | ||
| + | 2 µl BSA | ||
| + | 15 µl DNA | ||
| + | 0.5 µl EcoRI | ||
| + | 0.5 µl HindIII | ||
| + | 2) Run digestion for 90 minutes at 37ºC | ||
| + | |||
| + | Clean up: | ||
| + | 1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl | ||
| + | |||
| + | |||
| + | Plasmid digestion: | ||
| + | 1) Use miniprepped plasmid | ||
| + | 2) Prepare plasmid digestion reaction | ||
Revision as of 15:37, 20 July 2007
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
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:
Note that PCR product and vector digestion can be run simultaneously
PCR product digestion: 1) Prepare 20 µl digestion reaction: 2 µl EcoRI buffer 2 µl BSA 15 µl DNA 0.5 µl EcoRI 0.5 µl HindIII 2) Run digestion for 90 minutes at 37ºC
Clean up: 1) Qiagen PCR cleanup kit (see page 3 for protocol) and elute with 35 µl instead of 50 µl
Plasmid digestion:
1) Use miniprepped plasmid
2) Prepare plasmid digestion reaction
