Swartz:Protocols/Restriction Cloning

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.)