Swartz:Protocols/Restriction Cloning
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currently this page https://docs.google.com/document/d/1msML32vGmNI5klFypOuzPOZh5yB5MlatP7IZytZF1lQ/edit And https://docs.google.com/document/d/11cG_RlyzdjwNPZym6uRUbUybrXmqAmthMlhTHvyH5y8/edit
Vector Preparation
- Digest miniprepped vector with appropriate restriction enzymes for an appropriate time at 37C
- -ex)
- 41 uL pY71 CAT
- 5uL NEB 4
- 2uL NdeI
- 2uL AatII
- -consider diluting vector with water if using more than a few micrograms of DNA
- -aim for ~4h or so for digestion
- -heat inactivate enzymes for 30min at 80C
- Gel purify entire contents
- Measure final concentration using Qubit
Insert preparation
- Run appropriate PCR reactions to obtain full length gene products
- -may entail several annealing and amplification cycles for fusion products
- Gel purify full-length product (You can get away with a PCR purification if the product is very pure.)
- Measure concentrations using Qubit
- Digest with appropriate restriction enzymes for an appropriate time at 37C
- -ex)
- 30uL insert DNA
- 4uL NEB 4
- 2uL NdeI
- 2uL AatII
- -aim for ~4hr or so for digestion
- -heat inactivate enzymes for 30min at 80C
Ligation and Transformation:
See DNA_ligation for more details.
- Use 20ng of vector and a 3:1 molar ration of insert: vector for 20uL ligations with T4 ligase
- -ex)
- 4uL 5ng/ul pY71 vector (~1.7kb)
- 2uL 10ng/ul insert of 600bp (same mass added; since insert is ~1/3 size of vector, it evens out with 3:1 molar ratio)
- 2uL T4 ligase buffer
- 1uL T4 ligase
- 11uL water
- Ligate on benchtop for 2h
See Bacterial_transformation for more details
- Transform 5uL of ligation into 50uL chemically competent cells
- -place on ice 30min
- Heat shock for 45sec at 42C
- Place back on ice for 5min
- Add 500uL SOC
- -shake at 37C, 1h
- Plate entire mixture on appropriate antibiotic resistance plate (ie Kanamycin for pY71) at 37C
- -colonies should begin to form at ~12h
Tips
- (If I get no colonies, the first thing I usually do is replace the T4 ligase. When I try again, I’ll also use a few different insert ratios in addition to 3:1. I try to be sure that the vector is digested properly before beginning any cloning.)
