Matt Gethers/CRI, Thailand/Labwork/Ligation

=Ligations=


 * Make the following reaction mix:


 * Incubate at room temperature for 2 hours.

6.20.08
Ran 3 ligations - HmgR ORF cut with NdeI and BamHI ligated into pET-11a cut with the same; HmgA downstream fragment (1) blunted ligated into pUC18 at SmaI locus (cut with SmaI then blunted); HmgR upstream fragment (3) cut with PstI ligated into pUC18 cut with the same. The blunt end ligation got 2 &mu;l PEG 4000 while the other two reactions got H20 in place of the PEG. Incubating for 2 hours at room temp as per protocol.

6.25.08
Ran 2 ligations - HmgR ORF cut with NdeI and BamHI ligated into pET-11a cut with the same and the HmgR upstream fragment (3) cut with PstI ligated into pUC18 cut with the same. I didn't use PEG because neither of these are blunt end ligations, but I realize that I used too much ligase (should be 0.2-0.4 &mu;l, but I used 1 &mu;l because that's what the blunt end calls for). I'll let the reaction sit at room temperature for 2 hours, started 1145 and will end 1345 (It turns out I let it sit at room temp for about 3 hours). I had a higher insert/vector ratio this time - 2.5 &mu;l vector and 10 &mu;l insert. Depending on how my afternoon looks, I'll repeat the transformations tonight - actually, I may wait until I get a credible result from the PCR on the pET-11a/HmgR transformation before I try the transformations again.

7.4.08
I ligated the HmgA upstream fragment (2 by PiSo's system) into pKn001.P1 cut with HincII and blunted with End-It to produce pKn003.L1. I used the blunt end protocol (add 2 &mu;l of PEG), used only 2.5 &mu;l of vector, and 2.5 &mu;l of water for this ligation.

I also ligated the HmgR downstream fragment (4 by PiSo's system) into pKn002.P1 cut with SphI and HindIII to produce pKn004.L1. I followed the sticky end protocol (no PEG added), used only 2.5 &mu;l of vector and 4.5 &mu;l of water for this ligation.

Both incubated at room temp for two hours from 1530 to 1730 and then were placed in the -20.

7.14.08
I ligated the HmgA upstream fragment (2 by PiSo's system) into pKn001.P1 cut with HincII (7.10.08) and blunted with End-It (7.10.08) to produce pKn003.L2. I used the blunt end ligation protocol (add 2 &mu;l of PEG), used only 2.5 &mu;l of vector, 10 &mu;l insert, and 2.5 &mu;l of water for this ligation.

I also ligated the HmgR downstream fragment (4 by PiSo's system) into pKn002.P1 cut with SphI and HindIII (7.10.08) to produce pKn004.L2. I followed the sticky end protocol (no PEG added), used only 2.5 &mu;l of vector, 10 &mu;l insert, and 4.5 &mu;l of water for this ligation.

I also did two control ligations - one with pKn001 cut with HincII ligated to itself (2.5 &mu;l vector, no insert, 12.5 &mu;l water, 2 &mu;l PEG), and one with pKn002 cut with SphI-HindIII ligated to itself (2.5 &mu;l vector, no insert, 12 &mu;l water).

Both incubated at room temp for three hours and then were placed in the -20.

7.22.08
I treated half of the pKn002 SphI/HindIII digest (7.21.08) with a phosphatase - I'm hoping to reduce my background if I'm successfully doubly digesting any of the vector. I'm going to ligate fragment 4 into both of these vectors to produce pKn004.L3 and pKn004.L4. 2 &mu;l vector, 4 &mu;l fragment, 2 &mu;l buffer, 1 &mu;l ligase, 11 &mu;l water. I'm also going to ligate over night - hopefully the product will yield something in the transformation!

7.24.08
I'm ligating freshly digested HmgR ORF into digested pET-11a (same link, 6.17.08) to produce pIs001.L3. Using volumes as written; no PEG, so 2 &mu;l water. Incubation for 2 hours at room temperature.

7.30.08
I ligated the and doubly digested and blunt-ended Lox/Gentamicin cassette into the BamHI digested and blunt-ended pKn003 and SphI digested and blunt-ended pKn004 to form pKn005.L1 and pKn006.L1 respectively. I used 2.5 &mu;l of each vector, 10 &mu;l of insert, 2.5 &mu;l of water, 2 &mu;l of PEG, and the other reagents as written. Incubated at room temp for ~2.5 hours before use in transformation.

8.1.08
I ligated the and doubly digested and blunt-ended Lox/Gentamicin cassette into the BamHI digested and blunt-ended pKn003 and SphI digested and blunt-ended pKn004 to form pKn005.L2 and pKn006.L2 respectively. Because my last transformation was so inefficient, I'm trying a higher insert/vector ratio. I used 2 &mu;l of each vector, 12.5 &mu;l insert, no water, and the other reagents as written. Will allow longer incubation at room temp: started at 1000, will end sometime after 1400.

8.2.08
Ligated freshly amplified and digested HmgR ORF into both freshly digested pET-11a and old pET-11a (6/17/08). Made master mix of 9 &mu;l water, 4 &mu;l buffer, and 2 &mu;l ligase, split into two 7.5 &mu;l aliquots, then added 10 &mu;l ORF to both and 2.5 &mu;l of each vector to the separate tubes. pIs001.L4: New vector; pIs001.L5: Old vector. On bench top at 1545 - will allow to go until 1745.

8.5.08
Ligated freshly amplified (8.2.08) and digested HmgR ORF into both freshly digested pET-11a and old pET-11a (digested 6/17/08). Made master mix of 10 &mu;l water, 4 &mu;l buffer, 2 &mu;l ligase, and 20 &mu;l HmgR ORF. Split into two 18 &mu;l aliquots, then added 2 &mu;l of each vector to the separate tubes. pIs001.L6: New vector; pIs001.L7: Old vector. Incubated for 5 hours from 1210 to 1710 at room temperature.

8.6.08
Ligating freshly digested HmgR ORF (but not run out on gel or PCR clean up column) into digested pET-11a (8.5.08). 2.5 &mu;l vector, 10 &mu;l HmgR ORF, 7.5 &mu;l water, 2 &mu;l buffer, 1 &mu;l ligase. Incubated for 2 hours at room temperature.

8.12.08
I ligated the and doubly digested and blunt-ended Lox/Gentamicin cassette into the BamHI and XbaI digested and blunt-ended pKn003 to form pKn005.L3 and L4 respectively. I'm trying an even higher insert/vector ratio. I used 1 &mu;l of each vector, 16 &mu;l insert, no water, no PEG, and the other reagents as written. Made a master mix of 32 &mu;l insert, 4 &mu;l buffer, and 2 &mu;l ligase. Split into two 19 &mu;l aliquots and added 1 &mu;l BamHI digest to the pKn005.L3 ligation and 1 &mu;l XbaI digest to the pKn005.L4 ligation. Will allow incubation at room temp for ~6 hours.