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Seventeenth week (10/16/2008)

We made a ligation reaction.
prepare two tubes, one with H2O, Buffer, vector, insert and ligase, and the other the same but without the insert, this will serve to verify that the two ends of the vector are not back together. We leave in a water bath at 26 ° C for one hour to correctly insert is linked to the vector. We then proceeded with the following protocol: Transformation of E.
coli
1. Add 100 ml of competent cells and 20 ml of ligation rx a sterile Eppendorf. Make control with 100 ml of competent cells and 0.5 ml of control plasmid (Qiagen kit purified x)
2. Incubate 30 minutes on ice
3. Incubate 2 minutes to 42 ˚ C
4. Incubate 5 min on ice
5. Add 1 ml (or 450 ml) of LB medium without ampicillin
6. Incubate 1 hour at 37 ˚ C to 225 rpm
7. Centrifuge cells and resuspend the pellet in remaining supernatant
8. Sowing the entire contents of each tube on plates with LB medium + AmpicilinaColocar in oven at 37 ˚ C

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Sixteenth week (02/10/2008)

We PCR to amplify the insert (800 bp promoter UGA3), and cut with restruccion enzymes (EcoRI and Bami .) When we add vector tube and let phosphatase in oven at 37 º C for an hour and a half to be desfosfate well. After three buffers purify the vector and insert.
prepare an agarose gel where we put the inserts and let it run.

We

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Fifteenth week (09/25/2008)

transformations again two weeks ago, as some had fungal or other contamination.
done likewise;
strains grew on rich medium plates, clean, centrifuged, use a solution that helps the plasmid into the cell, incubated and plated .

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Fourteenth week (18/09/2008)

note the results of the previous week. We used the plasmid
to purify one of the weeks and cut with a restriction enzyme ( HindIII .) We did it twice.
This is called digestion of a plasmid . It uses a buffer specific enzymes, the plasmid and filled with water. Then placed at 37 ° C during the period of one hour and a half.
In agarose gel 8% m / v ran the undigested plasmid at different concentrations and the two plasmids also digested in different concentrations.

The image shown in the first two wells the undigested plasmids (the two bands observed belong to the two ways in which this CCC (predominant) and OC (one of the chains broke leaving plasmid in a relaxed state). The following wells show the once digested plasmid at different concentrations, where you see a band that belongs to the state of linear plasmid (cut the two strings). At the end is the marker and use it to measure the amount of base pairs so make sure everything ran correctly.