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

Contents

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.)
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