Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 7.20.08

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7.24.08

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.

To Do:

  • 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

Summary:

Completed all three tasks today, notes are in each protocol. Hopefully I will have some colonies to screen tomorrow.

7.25.08

To Do:

Summary:

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.

7.26.08

To Do:

  • Make glycerols and preps of the pIs001.L3 colonies.
  • Transform pIs001.P4 and P5 (name of each of the preps) into BL21 DE3.

Summary:

I made glycerols of both colonies and did minipreps; I used the prep to transform BL21. Plates in incubator at 1700.

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