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 1μg pSB4K5 with N.BstNBI in NEB buffer 3 for 1hr at 55°C. Heat inactivated N.BstNBI at 80°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°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°C.
8. Froze ligations at –20°C for a day
9. 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.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°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°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?