Bacteria Transformation: Difference between revisions
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(New page: IGEM:MIT/2009/OfficialWiki Bacterial Transformation 1. Take and aliquot of RbCl2 competent cells and thaw on ice 2. Add 10ul of Ligase Reaction 3. Incubate on ice for 30 min 4. He...) |
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[[IGEM:MIT/2009/OfficialWiki]] | [[IGEM:MIT/2009/OfficialWiki]] | ||
Bacterial Transformation | '''Bacterial Transformation''' | ||
1. Take and aliquot of RbCl2 competent cells and thaw on ice | 1. Take and aliquot of RbCl2 competent cells and thaw on ice | ||
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9. Grow at 37C till culture is cloudy | 9. Grow at 37C till culture is cloudy | ||
Mini-Prep (See QIAgen Kit Protocol) | '''Mini-Prep (See QIAgen Kit Protocol)''' | ||
1. After the mini-prep digest 1ul of plasmid | 1. After the mini-prep digest 1ul of plasmid | ||
'''20u Digestion:''' | |||
1ul Plasmid | 1ul Plasmid | ||
2ul of NE Buffer | |||
2ul of NE Buffer | (2ul 10x BSA) | ||
2ul 10x BSA | |||
0.5ul of Restriction Enzymes | 0.5ul of Restriction Enzymes | ||
Remaining ul are H20 | |||
2. Digest the Plasmid for 1 hour | 2. Digest the Plasmid for 1 hour | ||
3. Run on gel to determine whether the insert has been incorporated | 3. Run on gel to determine whether the insert has been incorporated | ||
Revision as of 13:02, 28 July 2009
Bacterial Transformation
1. Take and aliquot of RbCl2 competent cells and thaw on ice
2. Add 10ul of Ligase Reaction
3. Incubate on ice for 30 min
4. Heat shock at 42 for 30s, move immediately to ice
5. Add 0.5ml of pre-TB
6. Shake for 30 min at 36C
7. Plate on LB/Amp grow in 37C overnight
8. Add one colony to 2ml of TB/Amp Liquid Culture
9. Grow at 37C till culture is cloudy
Mini-Prep (See QIAgen Kit Protocol)
1. After the mini-prep digest 1ul of plasmid
20u Digestion: 1ul Plasmid 2ul of NE Buffer (2ul 10x BSA) 0.5ul of Restriction Enzymes
Remaining ul are H20
2. Digest the Plasmid for 1 hour
3. Run on gel to determine whether the insert has been incorporated
