Matt Gethers/CRI, Thailand/Labwork/Isolating HmgR/Week of 8.3.08
- Make colony suspensions of 8.2.08 transformants and inoculate cultures for prep tonight.
- Start colony PCRs to verify that the HmgR ORF is present.
Started four 3 ml cultures from each transformation (pIs001.L4 and L5) at 1000. Master Mix: 25 ml LB, 25 μl Amp, split into 8, add 25 μl appropriate suspension. Will return this evening to prep and transform into BL21 DE3.
All colony PCRs failed. I didn't include a positive control, so I can't be sure the result is correct. Seeing as how digested pET-11a shouldn't be able to religate to itself and that I did a control ligation with previously digested pET-11a that has worked before, I'm hoping there was something wrong with the PCR and will repeat it tomorrow. Otherwise, I might have not achieved complete digestion.
- Select eight fresh colonies from 8.2.08 pIs001.L4 and pIs001.L5 transformation plates and repeat colony PCRs.
- If products show up, inoculate cultures this afternoon to prep and transform into DE3 on Tuesday.
- If nothing shows up, repeat digest of vector - perhaps longer.
Colony PCRs failed and positive control worked. The HmgR ORF is not present. I need to do the cloning over again.
- Get more pET-11a (I don't remember who I got it from).
- Digest with nothing, NdeI, BamHI, and both to ensure the enzymes are cutting properly.
- Digest longer than last time - at least an hour.
- If digest is occurring properly, vector really shouldn't be able to recircularize, especially after removing the small fragment between NdeI and BamHI via a gel.
- Digest more HmgR ORF that I amplified over the weekend. Digest both with NdeI and BamHI. Diagnostic digest won't help because the digested fragment is little different from the undigested fragment, but run out on gel anyway.
- Ligate the fragments into each other. Use the old vector again as a negative control for induction.
- Ligate a bit longer this time as well, maybe 3 or 4 hours.
- Transform into DH5α.
Note: The previously digested pET-11a has worked during ligation before, so seeing as how it behaved the same way as the newly digested vector, I'm thinking the problem is downstream of the digest - probably the ligation.
Got more pET-11a from p'Koy and digested both pET-11a and HmgR ORF with NdeI and BamHI along with some control digests. Ran out on gel and purified. Used in ligation including both newly digested pET-11a and previously digested pET-11a. Transformed into DH5α.
- Start cultures of pIs001.L6/L7 transformants, do colony PCRs.
- This evening, make glycerols of good candidates and prep.
- Transform prep into BL21 DE3.
Made ten 3 ml cultures of LB amp. 8 were for pIs001.L6 and 2 were for pIs001.L7 transformants. Made a master of 30 ml LB, 30 μl amp. Aliquotted 3 ml to each of the 10 tubes. Used 10 μl of colony suspension (1 colony in 25 μl water) to inoculate each culture. In at 0900. Colony PCRs showed no colonies again. After looking/thinking back to the original cloning, I realize that I didn't run the HmgR ORF out on a gel or on a PCR clean up column. I think I may be losing a lot in the process - may cause my cloning to be inefficient. I digested some left over HmgR ORF Amplicon (8.2.08) with NdeI and BamHI and will only inactivate - no gel, no column. I ligated this digest into previously digested pET-11a (digested 8.5.08) and let run for 2 hours at room temp. Used this ligation product (pIs001.L8) to transform into DH5 alpha.
- Grow up cultures of new transformants and do colony PCR.
- If any good candidates, make glycerols, prep and transform into DE3.
I got a positive colony PCR result, so I think increasing the concentration of HmgR ORF in the ligation did the trick. I was doing FA late in the evening, so I didn't prep and transform into BL21 DE3. I'll start cultures tomorrow morning and try to prep and transform before I leave.
- Grow up cultures of 8.6.08 transformation colonies 2 and 3.
- Make glycerols, prep, and transform into DE3.
Grew up two 3 ml cultures using 8.6.08 transformation colonies 2 and 3 (6 ml master, 6 μl Amp, split in two, 10 μl colony suspension added to each), in at 0730, out at 1930. Made glycerols (put in wing C -80 because wing A locked) (1 ml culture, 500 μl 45% glycerol) and did a miniprep with the remaining cultures to form pIs001.P7 and pIs001.P8. Transformed 1 μl of each into BL21 DE3 - quick and dirty. Plates in at 2100.
- Do a mini isolation protocol for HmgR with the pIs001.P7 and P8 DE3s.
- If I get induction, first rejoice, then start an overnight to prepare for a large isolation protocol tomorrow.
Induction failed. Upon reviewing sequencing from pIs001.P4 and P5, I see that the ORF wasn't present in the plasmid. It's likely that the same thing happened here (P7 and P8). I'll talk with Mayuree on Monday about what's going wrong.