IGEM:UNAM-Genomics-Mexico/2009/Notebook/iGEM 2011 UNAM-Genomics Mexico/2011/05/23

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PBBRMCS-5 Digestion, Blunting and Ligation with PSB1T3

Today PBBRMCS-5 Plasmid was digested with Hind III and XBA I.

It's extremes were blunted and then the plasmid was cleaned.

It was dephosphorilated and then ligated to the PCR product of PSB1T3.

PBBRMCS-5 Plasmid Disgestion

PBBRMCS-5 was digested with HIND III and XBA I enzymes from NEB.

  • 5 μL of NEBuffer 2
  • 0.5 μL of BSA
  • 1.5 μL of XBA I
  • 1.5 μL of HIND III
  • 44.5 μL of Plasmid DNA

For a total reaction of 50μL. It was digested for an hour and a half with each of the enzymes at 37ºC.

Electrophoresis Gel

  • 1st Lane -> 5 μL Ladder (Fermentas)
  • 2nd Lane -> 5 μL Plasmid PBBRMCS-5 undigested, 2 μL DNA dye (Loading dye)and 5 μL of mq water.
  • 3rd Lane -> 5 μL Plasmid PBBRMCS-5 digested with XBA I, 2 μL DNA dye (Loading dye)and 5 μL of mq water.
  • 4th Lane -> 5 μL Plasmid PBBRMCS-5 digested with XBA I and HIND III, 2 μL DNA dye (Loading dye)and 5 μL of mq water.

The gel ran for 110 min at 90 volts.

Blunting Extremes

  • 0.6 μL of Blunting Buffer 10X
  • 2.5 μL of dNTP mix (1mM)
  • 19 μL of Digested Plasmid DNA
  • 1 μL of Blunt Enzyme (T4 DNA polymerase)
  • 1.9 μL of H20

for a total of 2 reactions of 25 μL each. Incubate for 15 min at 12ºC.

Inactivate with 1 μL of EDTA 0.5 mM and 24 μL of mq water.

The result of the blunting was cleaned using Roche's High Pure PCR Product Purification Kit.

The Plasmid PBBRMCS-5 DNA and the PCR product (Plasmid PSB1T3) is quantified by Nanodrop.

The result was 3 ng/μL

Dephosphate with Antarctic Phosphatase

  • 26 μL of Plasmid PBBRMCS-5 DNA
  • 3 μL of Antarctic Phosphatase Buffer
  • 1 μL of Antarctic Phosphatase enzyme

Incubate for 20 min. at 37ºC. Inactivate for 5 min. at 65ºC,

Ligation of PBBRMCS-5 with PSB1T3

  • 4 μL of PCR amplification product (insert)
  • 13 μL of Plasmid PBBRMCS-5 (vector)
  • 2 μL of Ligase Buffer
  • 1 μL of T4 DNA ligase


Controls
  1. Digested Vector without dephosphating and insert
  2. Digested and dephosphated vector without insert
  3. Vector with insert

Leave overnight at room temperature and check in the morning.


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