Knight:TOPO vector preparation: Difference between revisions

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Do a purification step? Or use it directly?
Do a purification step? Or use it directly?
Some TOPO cloning tries on pSB4K5.
I '''bolded''' the parts that change from the first to the second try.
1/22/08
1. Nicked 1µg pSB4K5 with N.BstNBI in NEB buffer 3 for 1hr at 55*C. Heat inactivated N.BstNBI at 80*C for 20min.
2. Purified half of nicked 4K5 with Quiagen PCR purification kit.
3. Suspended purified 4K5 in 1x Unit Test Definition (UTD) buffer (above), kept other half in NEB3.
4. Bound 10units TopoisomeraseI from Epicentre Biotechnologies to 4K5 in UTD buffer and NEB3 for 1hr in 37*C.
5. Got PCR product from Invitrogen PCR Supermix, prefix and suffix primers, and RFP reporter cassette. Gels showed right size bands, plenty of DNA.
6. Purified all of PCR product with Quiagen PCR pur. Kit.
7. Added 55ng, 110ng, and 220ng PCR product to 40ng of 4K5 in UTD buffer and 50ng 4K5 in NEB3 (so 6 tubes in all, plus empty backbone controls); ligated for 1hr at 37*C.
froze ligations at –20*C for a day
8.  Transformed into Top10 cells and plated on LB+Kan(40µg/ml).
No colonies above background.
1/28/08
1. Nicked 1µg pSB4K5 with N.BstNBI in NEB buffer 3 for '''2hrs''' at 55*C; '''did NOT heat inactivate N.BstBNI.'''
2. Purified half of nicked 4K5 with Quiagen PCR purification kit.
3. Suspended purified 4K5 in 1x Unit Test Definition (UTD) buffer (above), kept other half in NEB3.
4. Bound 10units TopoisomeraseI from Epicentre Biotechnologies to 4K5 in each UTD buffer and NEB3 for 1hr in 37*C.
5. Got PCR product from Invitrogen PCR Supermix, prefix and suffix primers, and RFP reporter cassette. Gels showed right size bands, plenty of DNA.
'''6.''' '''Purified half of PCR product with Quiagen PCR pur. Kit, kept other half in supermix buffer.'''
7. '''Mixed 1:1, 1:2, 1:5 ng 4K5:PCR product for each 4K5 and PCR product in different buffer (so in the end had 12 tubes: 2x4K5 buffers, 2x PCR product buffers, 3x concentrations of backbone:insert). Ligated for 15min at room temp, 15 min at 37*C (15min RT is what the Invitrogen TOPO cloning kit uses, so I thought I’d try it).'''
8.  '''Immediately''' transformed into Top10 cells and plated on LB+Kan(40µg/ml).
No colonies above background.
Any suggestions?


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Latest revision as of 10:01, 1 February 2008

Overview

This is a made-up protocol to prepare pSB**5 vectors for TOPO cloning. It has not been verified at all!

Materials

Procedure

Nick plasmid

  1. Mix
    • X μL plasmid
    • 1 μL Nt.BstNB I
    • 5 μL NEBuffer 3
    • 44-X μL H2O
  2. Incubate at 55°C for at least 1 hour
  3. Heat-inactivate at 80°C for 20 mins

Do a purification step? Try the same buffer?

Covalently attach DNA topoisomerase I from Vaccinia

Quality control procedure

  1. Mix a 50 μL reaction including
    • 50 mM Tris-acetate (pH7.5)
    • 100 mM NaCl
    • 2.5 mM MgCl2
    • 0.1 mM EDTA
    • 1 μL DNA topoisomerase I from Vaccinia
  2. Incubate at 37°C for at least 1 hour

Unit test definition

  1. Mix
    • 40 mM Tris-acetate (pH 8.3)
    • 100 mM NaCl
    • 2.5 mM MgCl2
    • 1 mM EDTA
    • 1 unit of DNA topoisomerase I from Vaccinia
  2. Incubate at 37°C for 1 hour

Alternative protocol from Shuman et al.

From Shuman 1994: maximum topoisomerase activity was observed with a 20 μL reaction mixture containing

  • 50 mM Tris-HCl (pH 8.0)
  • 100 mM NaCl
  • 5 mM MgCl2
  • 5 mM ATP
  • 0.3 μg of plasmid DNA
  • 0.63ng of Vaccinia topoisomerase I, incubated at 37°C for five minutes.

Do a purification step? Or use it directly?


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