Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 6.22.08
- Select 6-7 colonies from HmgR-pET-11a transformation and make colony suspensions.
- Use part of colony suspension for PCR.
- For colonie(s) that have screened properly, inoculate culture(s) to make glycerol(s) (maybe do this step overnight).
Made 50 μl colony suspensions and used 5.1 μl in PCRs. Ran the PCR used to amplify the HmgR ORF to screen for transformants. Here's the gel info. No products, but the positive control is not right (way too long). I'm going to run a PCR using PFU to screen the colonies and also to compare the positive control with the 2x supermix.
- Run PCR products from PFU screening out on gel - see if anything shows up and how the positive control from PFU compares with the same from the 2x supermix.
- If PFU positive control is OK and no products - repeat ligation. If the 2x supermix is still weird, I might just avoid using it for this screening.
- If PFU positive control is OK and some products - streak out new plate from suspension to get single colonies.
- Run a second set of screening for the pet-lla-HmgR Orf ligation
- Ask Sopapan what enzyme I should use for the screening if not the supermix.
- Maybe I should include another run of colony 5 just to be sure I didn't make a mistake (it was the colony immediately proximate to the positive control...)
Ran the products out on a gel, looks like the supermix is acting weird. The Pfu positive control worked properly while the supermix did not. Additionally, it seems that colony five has the correct product, so I've streaked out a plate from 5 μl of the suspension and placed it in the 37oC incubator. I'll try to get a single colony tomorrow. I also started another round of PCR screening from the pET-11a/HmgR transformants. I inoculated 5 colonies in 50 μl H20 each. Later, I'll need to grow up a culture and mini prep.
- Select two single colonies from the plate streaked from colony 5 of pet-11a-HmgR ORF transformation and start cultures.
- Part of the cultures will be for mini prep, the other for glycerol.
- Run the colony PCR products out on a gel to see if anything else has the plasmid with insert and to verify that colony 5 has the insert.
Over the next few days, I need to grow up a culture of the putative transformant (pET-11a/HmgR in DH5-α) to make both a glycerol and to miniprep. With the prep, I need to send it to be sequenced. That should be submitted before Friday so it has time to process. Simultaneously or after the sequencing comes back, I can transform the plasmid into BL21 DE3 for expression. These transformants aren't very stable, so I think I'll have to transform anew every time I want to isolate the HmgR protein. I'm not sure if I make a glycerol of any of the transformants (for what reason I'm not sure) or if I just inoculate a culture straight away. Inoculate 1 or 2 cultures in LB Amp (150-200 μg/ml) and 0.4% glucose to repress expression. Grow up the culture to between OD 0.4 and 0.8, then split into two. Add IPTG to one of the two cultures to 1 mM. Grow for 2 hours (cells should be approximately the same density after so short a time). Spin down the cells and resuspend in ~300 μl 50 mM phosphate buffer. Sonicate the cells to rupture. Spin down the lysed cells at 10,000 RPM (how long?). Remove the supernatant and hold onto it. Wash the pellet in 50 mM phosphate buffer, spin down again, and resuspend in 300μl PB one more time. At this point, I should do a Bradford assay to determine approximately how concentrated my samples are. I want to load approximately the same amount of protein in each lane of the poly-ac gel (SDS-PAGE, I think). Load the poly-ac gel with a protein marker (prestained?) and look for protein at the appropriate length (have to figure out what the length is). After confirming the expression and length of the protein, I'll try to purify it via a heparin column (more on that later).
I set up an additional PCR to screen for more pET-11a/HmgR transformants (also PCRing the pUC18/HmgA Downstream). This PCR uses Taq rather than the supermix because the latter just isn't working for me (see gel info here). In this third attempt, I forgot to include the original colony #5 to retest if a band does show up. That's OK. I'm just going to trust that I didn't make a mistake and go ahead with the culture and mini prep. I inoculated two 5 ml cultures from the colony 5 streaked plate (streaked from colony suspension yesterday); LB+Amp (1/1000). In the 37oC shaker at 1700.
- Run the Taq-PCR out on a gel and see if any more of the pET-11a/HmgR colonies are correct.
- If any products show up, inoculate a culture this evening and mini prep tomorrow.
- If no products show up, talk with Mayuree about screening more colonies.
- Mini-prep the colony 5 cultures and store in -20.
I ran out the colony PCR products along with a positive control on a gel and found that none of the colonies have product. I've screened 10 colonies now with only one candidate (colony 5). I should probably go ahead and repeat the transformation using yesterday's ligation I did with a higher insert/vector ratio. I made minipreps of the two colonies I inoculated yesterday afternoon as well as glycerols (1 ml culture, 500 μl 45% glycerol). The preps are in my -20 and the glycerols are in the -80 in wing A under Duan 2 in box "strains from others".
I'm repeating the PCR on the colony 5 suspension along with a positive control to be sure I'll be sending something out for sequencing.
- Run out the PCR product (to be sure that colony 5 has product) along with positive control on a gel.
- Run out ligation product for second round of pET-11a/HmgR on gel with controls.
- Transform pET-11a/HmgR ligation product into DH5-α and the pET-11a/HmgR miniprep into BL21 DE3.
- Ask Mayuree about getting into lab on Saturday to pick up transformants.
- Submit pET-11a/HmgR prepped plasmid for sequencing.
- Ask Mayuree how to submit sequencing
I ran out the PCR Product along with the positive control on a gel. Neither the colony PCR nor the positive control worked; I think the annealing temp/extension time was optimized for Pfu, so I may have to play around with it again. I'm going to have another batch of colonies to screen, so it's worth the effort to optimize the PCR. I submitted the colony 5 prep for sequencing. I transformed the prep from colony 5 into BL21 DE3 and I also transformed a fresh pET-11a/HmgR ligation product into DH5-α. Next week, I hope to start the purification process for HmgR and hopefully the sequencing will come back confirming that the ORF made it into the pET vector properly.
The transformation yielded colonies both in BL21 DE3 and DH5α. I'll put the former transformation in the 4 degree until Monday. I've been having trouble with the positive control in the PCR for the HmgR ORF (see 6.27.08 gel), so I need to optimize the annealing temp before I try the colony PCRs.
Schedule: Saturday morning: Set up PCR to optimize the annealing temp. Sunday evening: run gel to see where product shows up; set up colony PCRs at the appropriate temperature. Monday morning: run out products on gel and inoculate cultures for prep and glycerols.