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Yet More Ligation

Ligation Attempt #4

  • Removed both overnight digestions, the XbaI digestion of B0015 and the EcoRI and SpeI digestion of phsABC, from 7/8 from the incubator.
  • Heatkilled enzymes of phsABC digest with 20 minutes at 80°C.
  • Ran PCR purification protocol on XbaI digestion using microcentrifuge. In order to give a concentrated sample, eluted in only 35 μL of EB Buffer that had been heated at 50°C. Resulting concentration could not be determined as there was residual ethanol contamination.
  • Ran EcoRI digestion of purified XbaI digest product at 37 °C, with components as follows. Digestion was run for an hour before addition of CIP, after which it was run for another hour.
B0015 digestion with EcoRI
NEB EcoRI buffer 5 μL
BSA 100x 0.5 μL
NEB EcoRI 3.6 μL
B0015 digested with XbaI 25 μL
NEB Calf Intestinal Phosphatase 1 μL
Sterile H2O 15.9 μL

Followed with 20 minute heatkill at 80°C.

  • In order to set up ligation, need concentration of B0015, but ethanol contaminant continues to preclude measurement. Will estimate that the purification between digestion steps resulted in a 50% loss of DNA, leaving behind a 10 ng/μL concentration. Will also, as usual, run different ligation trials with different ratios of insert to vector, which should help combat any issues if this estimate is inaccurate.
  • Set up the following ligation trials, with stoichiometric ratios of insert to vector based on B0015 concentration estimate. Ran the ligations for 20 minutes at room temperature.
Ligation Reaction Components
Control Ligation 1:1 I:V 2:1 I:V 3:1 I:V 4:1 I:V
2 μL NEB T4 ligase buffer
1 μL NEB T4 ligase
5 μL B0015 solution
12 μL H2O 8.9 μL H2O 5.8 μL H2O 2.7 μL H2O --
-- 3.1 μL phsABC 6.2μL phsABC 9.3 μL phsABC 12 μL phsABC
  • Following ligation, transformed 1 μL of each ligation reaction into commercial grade cells according to standard transformation protocol. Also transformed triple ligation reaction.

Enzyme Activity Test

To test enzyme activity, will do single digests of B0015 with enzymes in question and then run them on a gel to see if the plasmid linearized. XbaI and EcoRI are old, so will test them first. For XbaI activity test, simply set aside 10 μL of digestion solution from overnight digestion. Testing of EcoRI required additional digests. Digests using both old and new EcoRI samples were run for two hours at 37 °C with the following components:

Gel of single digests of B0015 Left to Right lane 1:1 kb ladder, lane 2: uncut circular B0015, its dimer, and lane 3 spillover, lane 3: XbaI digest of B0015, lane 4: old EcoRI digest of B0015, lane 5: new EcoRI digest of B0015
EcoRI Activity Test
NEB EcoRI buffer 5 μL
BSA 100x 0.5 μL
NEB EcoRI 3.6 μL
undigested B0015 3.8 μL ( 1 μg DNA)
Sterile H2O 37.1 μL

Followed with 20 minute heatkill at 80°C.

  • Loaded all three enzyme activity tests on a 1.0% agarose gel and ran them at 90 V versus a 1 kb ladder and circular B0015 plasmid.

Based on the results, all enzymes appeared to work, as they successfully created a linearized 3.2 kb fragment.

Wetlab work for this day is also recorded on pages 54-57 of the hard copy lab notebook.