Glycerols preparation of the DH5α E.coli that carry Pamela Silver Lab's plasmids. Plasmid extraction procedure over these bacteria. Digestion with XbaI of these plasmids.
Today I prepared 3 eppendorf tubes with the DH5α E.coli carrying Pamela Silver Lab's plasmids. I used the little test tubes I left incubating yesterday. 500 μl were extracted from these tubes, no antibiotic was added as the LB liquid medium already had the appropiate antibiotic. Another 500 μl of glycerol at a concentration of 30% were added to the 500 μl taken from the test tubes. The total 1 ml volume was put into eppendorf tubes. They are supposed to be stored first into a -20°C fridge, and after ~12 hours passed into a -80°C fridge. Unfortunately, the revco that we have been used so far belongs to David Romero's lab; and nobody was at the lab to let me store the glycerols. I left them at one of Miguel's -20°C fridges.
I also extracted the transformed plasmids using Miguel's protocol. The extractions were added with 20 μl of TE-RNAse and left at the incubator for 30 minutes before doing the gel electrophoresis. The gel was performed during 35 minutes at 120 volts.
2.5 μl of DNA ladder was put into the first lane. In all the others wells, 3 μl of plasmid extraction along with 3 μl of dye were put.
Well #2 - pPS3182 (Alias D9). HydEF and HydG maturaction factors. Plasmid with Spectinomycin resistance cassette.
Well #3 - pPS3183 (Alias D23). PFOR. Plasmid with Chloramphenicol resistance cassette.
Well #4 - pPS3186 (Alias E237). HydA and Fd. Plasmid with Ampicilin resistance cassette.
Well #5 - Pablo García's PCR amplification product purified with Roche's High Purification PCR Products Kit.
A lot of chromosomal DNA contamination is observed. Regardless, the plasmids' isoforms are also observed. I proceeded performing a digestion of the plasmids to checked their sizes in another agarose gel. All the plasmids are devoid XbaI sites; however, each plasmid posses two multiple cloning sites. Each biobrick insertion adds one XbaI site (as this site is part of the Standard Biobrick prefix). Plasmid pPS3182 contains each of its genes into each multiple cloning site, same history for pPS3186; is is expected to see three bands in the gel for these two (assuming that the extracted plasmid DNA is purified, which is not). As pPS3183 only contains one gene, the plasmid contains only one XbaI site. The plasmid digestion was performed as following:
H20
4.75 μl
Buffer 4 10x
2.5 μl
BSA 0.1%
2.5 μl
DNA
14 μl
XbaI
1.25 μl
Total
25 μl
The digestions were left incubating at 37°C overnight.
UNAM Genomics Mexico 2011
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