Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 7.20.08
The sequencing of the two clones I have expressing HmgR is a little grim, so I need to try the cloning and purification of HmgR again, this time with a more reliable polymerase. Last night, I began two PCRs using HotStar. This morning, I ran a gel (link to from PCR page) and found that the PCR with buffer Q yielded product at the correct length, but not the reaction without buffer Q.
- Digest HmgR amplicon with NdeI and BamHI
- Ligate digested amplicon into pET-11a (should have some digested vector left over).
- Transform ligation product into DH5α
Completed all three tasks today, notes are in each protocol. Hopefully I will have some colonies to screen tomorrow.
- Select colonies from pIs001.L3 transformation and do colony PCRs.
- If any colonies run at the correct length, inoculate cultures this evening for glycerols and prep tomorrow.
Looks like all colonies I selected have the insert. I'm selecting colonies 2 and 3 for overnight cultures (colony 1 was a little faint, so I'm avoiding it). 5 ml culture, 5 μl amp, 25 μl colony suspension (made media in master mix x3). In at 1430.
- Make glycerols and preps of the pIs001.L3 colonies.
- Transform pIs001.P4 and P5 (name of each of the preps) into BL21 DE3.
I made glycerols of both colonies and did minipreps; I used the prep to transform BL21. Plates in incubator at 1700.