Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 6.29.08

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I streaked out 5 white-looking colonies from the pKn002.2 transformation on LB/Amp/X-gal/IPTG plates and placed them in the incubator overnight. Hopefully I can select colonies and do PCRs tomorrow morning.

1/1000 Amp (100 μl in 100 ml LB), 10 μl IPTG, 20 μl x-gal, 70 μl LB spread on each plate (made master mix x5).


To Do:

  • Prepare sequencing file to compare with sequencing data when it comes back.
  • Replace streaked plate in incubator for a few more hours.
    • If I can get single colonies from the plates, do colony PCRs. Otherwise, try streaking from these plates again (should have waited longer for IPTG/X-gal to dry).


When I initially removed the plates at 8 am, the colonies were small and they appeared to run together, but after 3-4 hours longer, I could definitely distinguish single white colonies. I picked the 7 most promising single white colonies of the four plates (I didn't use plate 2 because it got less IPTG and x-gal) and made colony suspensions. I used 1 μl of this suspension in a 10 μl colony PCR to determine the presence and directionality of the insert. I ran the products out on a gel and found that nothing (including the positive control) showed up. Given that white colonies are likely to have the insert, I suspect something went wrong with the PCR. I'll think about how to fix this tomorrow. Remember to record the information on the gel.


To Do:

  • Debug PCR for pKn002
  • Check sequencing data to see if pKn001 sequenced properly.
    • If it did, begin next cloning step.


After looking at the HmgR upstream fragment AFTER digestion with PstI, I realize that the BT2696 primer binding site is no longer present, so I never had a binding site for the PCR - that's why it failed. My options are BT1032 + M13 For or BT1033 + M13 Rev. I've successfully used both BT1032 and M13_for separately, however, the melting temperatures have a 10 degree differential while the other pair has only ~4.5 differential, so I'm going to go with M13_rev and BT1033 (I've successfully used 1033 before, so M13_rev is the only newcomer).

Tomorrow I need to revamp the PCR protocol and try again.

I also need to check the sequence data for pKn001.


To Do:

  • Revamp PCR protocol for pKn002.
  • Run PCR for pKn002
  • Check sequencing data to see if pKn001 sequenced properly.
    • Fix Vector NTI file for pKn001 (and 2 for that matter)
    • It's likely that I'll need to use another primer(s) to get more complete sequencing information.
    • If pKn001 sequenced properly, plan out and do next cloning step.


I retooled the pKn002 PCR protocol with different primers and it seems to have worked (see gel image here). I inoculated two 5 ml cultures (5 μl amp each) with 25 μl colony suspension from colonies 1 and 2 from the 6.27.08 pKn002.L2 (formerly known as pKn002.2) transformation. In at 1600.


To Do:

  • Submit pKn001.P2 (formerly know as pKn001.2) for sequencing with reverse primer (internal as well?).
  • Make glycerol and miniprep of pKn002 cultures.
  • Submit pKn002 for sequencing with forward and reverse primers.


I submitted pKn001.P2 (formerly known as pKn001.2) for sequencing with the pUC reverse primer. I also made glycerols and minipreps of both colonies transformed with pKn002.L2 (formerly known as pKn002.2) - the preps are pKn002.P1 and pKn002.P2 (formerly pKn002.3 and pKn002.4). I sent pKn002.P1 out for sequencing with pUC forward and reverse primers.

I can go ahead and drop the second fragments into pKn001 and pKn002 - hopefully the sequencing will come back ok. Be sure to save prep at each step.


To DO:

  • Digest pKn001.P1 with HincII and pKn002.P1 with SphI and HindIII.
  • Run the latter digest on an agarose gel and excise and purify the vector.
  • Use the End-It kit to blunt end the pKn001.P1 digest product.
  • Ligate the HmgA upstream fragment into pKn001.P1 to produce pKn003.L1 and ligate the HmgR downstream fragment into pKn002.P1 to produce pKn004.L1. (Note: from here on (pKn003/4 onward), my plasmid nomenclature will include an L or P after the decimal to denote a ligation product or prep respectively. The number after the L or P denotes the version of that product. I've renamed earlier products, but kept the old names as well so I don't confuse myself).


I digested both vectors with the appropriate enzymes and did the appropriate dressing afterwards (blunt ending or gel extraction and purification) and completed the ligations. I plan to transform on Sunday evening so I have colonies to work with on Monday morning. I'll need to develop PCR protocols to tell me whether or not I have good candidates - I don't think I have any way of differentiating between closed vectors the correct ligation product vis-a-vis blue-white selection.