Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 6.22.08
- 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.
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 μl), IPTG (10 μl), and x-gal (20 μl). 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.
- 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 μl in ~80 ml LB agar), and I spread IPTG (10 μl each plate) and x-gal (20 μl each plate). The IPTG and x-gal were added to LB (41, 82, and 287 μl respectively), then 100 μl aliquots were spread on each plate. Plates were placed in 37oC incubator at 10:30 AM.
I also made 1 μM stock of Primer 2696 which I'll need for the PCR to verify the presence and directionality of the HmgR upstream fragment.
- 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.
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.
- 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.
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.
- 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-α
Made glycerols of colonies 3 and 5, minipreped the same and submitted for sequencing. I also transformed HmgR Upstream Fragment/pUC18 into DH5-α.
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.