DNA purification and PCR

6-14-10
Using a 1% gel run for ~45mins at 110V, only the ladder was visible in imaging afterward. We speculate that this is because the nanodrop concentrations were so low that they cannot be visualized by this method. Therefore, we are using DNA purifcation followed by PCR to amplify the number of DNA products and thus allow them to be visualized to confirm their existence in these samples.
DNA purification:
Add entire sample (approx. 300ul) + 400ul of sterilized molecular biology grade H20 to filter/centrifuge tube. Centrifuge for 7 mins @3000rpm. Check the level of the liquid, continue process for additional 7mins @ 3000rpm if liquid not completely filtered through. Continue procedure until only film remains on filter. In this case, it was 7min, 7min, 5min. Then 50ul of water (sterile) was added to each filter. The filter was then added upside down to a 2mL centrifuge tube and gently agitated. Then each centrifuge tube was centrifuged at 3000rpm for 1 min.

PCR:
Master Mix:
H2O 224ul
10xbuffer 35ul
dNTP 28ul
27F 7ul
1492F 7ul
BSA 7ul
Taq Pol 7ul

45ul of master in each (sample 1, sample 1, sample 6, sample 6, DNA [+control], water [-control]) in addition to 5ul of template (sample). Total volume 50ul

6-15-10
PCR products were run on a 1% gel for ~40 mins. 5ul of each product was added to each lane in the order of +control, -control, Sample 1, Sample 1, Sample 6, Sample 6. 1kb ladder was first. Results showed that both PCR reactions were successful, indicating that there is in fact DNA in the samples collected. PCR will then be performed all the other samples (along with sample 6 again due to extremely low levels of DNA present in gel). The same procedure as the previous PCR will be used. Gel will be used to confirm this result

I also made 2L of LB Amp and poured them to make plates using the recipe provided in the "MEDIA" folder next to the weighing station.

6-16-10
Samples 5-9 are being subjected to PCR again, followed by a gel to confirm success of the PCR and that there is DNA in the samples. These samples are the only ones being PCRed again because they either did not work or were in extremely low concentrations post-PCR previously. The gel did not show any product and thus we are abandoning the samples from that site

PCR products 1,2,3, and 4 were de-salted using the procedure provided. It required a 7min, 7 min, 5 min spin

6-17-10
Using my PCR products 1-4, I performed a ligation using a procedure provided by a kit provided by my mentor.
Also, in the morning I began reading chapters assigned by my mentor.