Talk:Knight:TOPO vector preparation

Some TOPO cloning tries on pSB4K5.

I bolded the parts that change from the first to the second try.

1/22/08

 * 1) Nicked 1&mu;g pSB4K5 with N.BstNBI in NEB buffer 3 for 1hr at 55&deg;C. Heat inactivated N.BstNBI at 80&deg;C for 20 mins.
 * 2) Purified half of nicked pSB4K5 with Qiagen PCR purification kit.
 * 3) Suspended purified pSB4K5 in 1X Unit Test Definition (UTD) buffer (above), kept other half in NEB3.
 * 4) Bound 10 units TopoisomeraseI from Epicentre Biotechnologies to pSB4K5 in UTD buffer and NEB3 for 1hr in 37&deg;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) I purified all of PCR product with Qiagen PCR purification kit.
 * 7) Added 55ng, 110ng, and 220ng PCR product to 40ng of pSB4K5 in UTD buffer and 50ng pSB4K5 in NEB3 (so 6 tubes in all, plus empty backbone controls); ligated for 1hr at 37&deg;C.
 * 8) Froze ligations at –20&deg;C for a day
 * 9) Transformed into TOP10 cells and plated on LB+Kan(40&mu;g/ml).

No colonies above background.

1/28/08

 * 1) Nicked 1&mu;g pSB4K5 with N.BstNBI in NEB buffer 3 for 2hrs at 55&deg;C; did NOT heat inactivate N.BstNBI.
 * 2) Purified half of nicked pSB4K5 with Qiagen PCR purification kit.
 * 3) Suspended purified pSB4K5 in 1x Unit Test Definition (UTD) buffer (above), kept other half in NEB3.
 * 4) Bound 10 units TopoisomeraseI from Epicentre Biotechnologies to pSB4K5 in each UTD buffer and NEB3 for 1hr in 37&deg;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 Qiagen PCR purification kit. Kept other half in supermix buffer.
 * 7) Mixed 1:1, 1:2, 1:5 ng pSB4K5:PCR product for each pSB4K5 and PCR product in different buffer (so in the end had 12 tubes: 2xpSB4K5 buffers, 2x PCR product buffers, 3x concentrations of backbone:insert). Ligated for 15min at room temp, 15 min at 37&deg;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(40&mu;g/ml).

No colonies above background.

Any suggestions?