Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 6.22.08

=6.23.08=

To Do:


 * Look for any white colonies on plates in addition to what Mayuree found.
 * Streak out the white colonies on plates with Amp, IPTG, and x-gal - look for white colonies.

Summary:

Chose 4 white colonies that Mayuree found (3 from HmgA downstream and 1 from HmgR upstream) and streaked out on one plate (20 ml LB) with Amp (20 &mu;l), IPTG (10 &mu;l), and x-gal (20 &mu;l). The latter two reagents were spread on the plate rather than being included in the hot agar. Placed in 4 degree and will put in 37 degree incubator when I leave this evening.

I also made aliquots of M13 For and  M13 Rev so if and when I get single white colonies I can do a PCR both for the presence of the insert and the directionality for both the  downstream fragment of HmgA and the  upstream fragment of HmgR.

=6.24.08=

To Do:


 * Restreak colonies on plates (1 colony per plate). Place in incubator.

Summary: Picked single colonies from the plate I streaked yesterday. 2 colonies for the upstream HmgR fragment and 2 colonies for the downstream HmgA fragment. Each colony was streaked on its own plate; each plate had 1/1000 Amp (included 80 &mu;l in ~80 ml LB agar), and I spread IPTG (10 &mu;l each plate) and x-gal (20 &mu;l each plate). The IPTG and x-gal were added to LB (41, 82, and 287 &mu;l respectively), then 100 &mu;l aliquots were spread on each plate. Plates were placed in 37oC incubator at 10:30 AM.

I also made 1 &mu;M stock of Primer 2696 which I'll need for the  PCR to verify the presence and directionality of the HmgR upstream fragment.

=6.25.08=

To Do:


 * If I get any single white colonies from both the HmgA downstream and HmgR upstream fragment transformations, inoculate a few in H20 and do a colony PCR for the downstream fragment of HmgA and the upstream fragment of HmgR.
 * If the PCRs come back positive, I can start cultures for both miniprep and glycerols.

Summary:

I got white colonies on the Downstream HmgA fragment/pUC18 plates that I streaked out. I selected 6 of these and did a colony PCR using supermix, but it seems supermix isn't working quite right. I repeated the PCR in the evening alongside the pET-11a/HmgR colonies using a Taq kit from Finnzyme.

=6.26.08=

To Do:


 * Run the Taq-PCR products out on a gel to see if anything turns up.
 * If products, inoculate cultures with the colony suspensions this evening.
 * If no products, recheck protocol to be sure I haven't made any mistakes.

Summary:

Ran the colony PCR out on  a gel and found that 5 of 6 colonies I screened have the product with the correct directionality. It looks like the ligation, transformation, and Blue-White selection worked.

I'm inoculating 2 5 ml cultures (1/1000 Amp), one from "colony 3" suspension and one from "colony 5". These correspond to the 2nd and 4th lanes of the gel. I accidentally added loading dye to the "colony 1" suspension, so rather than renumbering everything, I just started with 2 and added colony 7 for a total of 6 colony suspensions.

Tomorrow I will try a second transformation of the HmgR upstream fragment into pUC18. I tried another ligation and I should run this out on a gel tomorrow just to see how efficient the ligation was.

=6.27.08=

To Do:


 * Make glycerols of colonies 3 and 5 that I grew up in culture yesterday evening.
 * Miniprep cultures 3 and 5.
 * Submit prep(s) for sequencing.
 * Ask Mayuree how to submit for sequencing.
 * Transform HmgR Upstream Fragment/pUC18 into DH5-&alpha;

Summary:

Made glycerols of colonies 3 and 5, minipreped the same and submitted for sequencing. I also transformed HmgR Upstream Fragment/pUC18 into DH5-&alpha;.

=6.28.08=

Got plates from yesterday's transformation, and it looks like there are a few white candidates, so I need to streak out some plates (to be sure I have single white colonies), and afterwards I can make suspensions and try colony PCRs.

Schedule: Sunday evening, streak out plates from the single colonies. Leave overnight. Monday morning, inoculate individual white colonies in water and do the appropriate colony PCR for pKn002.