Matt Gethers/CRI, Thailand/Labwork/Ligation

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Contents

Ligations

  • Make the following reaction mix:
Reagent Volume
Vector DNA5 ┬Ál (50-400 ng)
Insert DNA10 μl (use a 1:1 up to a 3:1 molar ratio of insert DNA termini to vector DNA)
10x ligation buffer2 μl
50% PEG 4000 (for blunt end only)2 μl
H200 (2 μl if not blunt end)
Ligase1 μl
Total20 μl
  • Incubate at room temperature for 2 hours.

Run Notes

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 μl 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 μl, but I used 1 μl 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 μl vector and 10 μl 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 μl of PEG), used only 2.5 μl of vector, and 2.5 μl 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 μl of vector and 4.5 μl 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 μl of PEG), used only 2.5 μl of vector, 10 μl insert, and 2.5 μl 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 μl of vector, 10 μl insert, and 4.5 μl of water for this ligation.

I also did two control ligations - one with pKn001 cut with HincII ligated to itself (2.5 μl vector, no insert, 12.5 μl water, 2 μl PEG), and one with pKn002 cut with SphI-HindIII ligated to itself (2.5 μl vector, no insert, 12 μl 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 μl vector, 4 μl fragment, 2 μl buffer, 1 μl ligase, 11 μl 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 μl 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 μl of each vector, 10 μl of insert, 2.5 μl of water, 2 μl 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 μl of each vector, 12.5 μl 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 μl water, 4 μl buffer, and 2 μl ligase, split into two 7.5 μl aliquots, then added 10 μl ORF to both and 2.5 μl 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 μl water, 4 μl buffer, 2 μl ligase, and 20 μl HmgR ORF. Split into two 18 μl aliquots, then added 2 μl 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 μl vector, 10 μl HmgR ORF, 7.5 μl water, 2 μl buffer, 1 μl 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 μl of each vector, 16 μl insert, no water, no PEG, and the other reagents as written. Made a master mix of 32 μl insert, 4 μl buffer, and 2 μl ligase. Split into two 19 μl aliquots and added 1 μl BamHI digest to the pKn005.L3 ligation and 1 μl XbaI digest to the pKn005.L4 ligation. Will allow incubation at room temp for ~6 hours.

References

Fermentas T4 DNA Ligase

Protocol for T4 DNA Ligase

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