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.
For a total reaction of 50μL. It was digested for an hour and a half with each of the enzymes at 37ºC.
The gel ran for 110 min at 90 volts.
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
Incubate for 20 min. at 37ºC. Inactivate for 5 min. at 65ºC,
Ligation of PBBRMCS-5 with PSB1T3
Leave overnight at room temperature and check in the morning.