Swartz:Protocols/Restriction Cloning: Difference between revisions
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https://docs.google.com/document/d/1msML32vGmNI5klFypOuzPOZh5yB5MlatP7IZytZF1lQ/edit | 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.) | |||
[[Category:Swartz_Protocols]] |
Latest revision as of 16:45, 28 May 2012
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.)