T3+Specific+Procedure

Genomic DNA from Plant User Manual:

DNA Extraction Procedure for Samples 26-30:
 * We followed the procedure in section 5.1, pages 15-18 in the above pdf, for standard protocol for genomic DNA from plant.
 * In step 1, we homogenized the sample by grinding with sand for a better homogenization. We then followed step 2a for cell lysis. In step 3, because there was no specific μL for the collection of the clear flow-through, we collected the accumulated μL in the tube which was 420 μL. (At this point in the procedure, Buffer PE should be placed in the water bath at 65 degrees Celsius for use in step 7.) In step 6 for the 2nd wash, we discarded the flow-through, centrifuged the Column, and the discarded the flow-through again to ensure that the ethanol in Buffer PW2 was removed from the filter.

Re-suspending Primers

PCR Procedure

The PCR solution will contain:
 * 1µL of 10µM forward primer
 * 1µL of 10µM reverse primer
 * 12µL nuclease free water
 * 1µL template DNA
 * 15µL of MasterMix (2x)

The PCR will run through:
 * 1) 1 minute @ 94°C
 * 2) 1 minute @ 94°C
 * 3) 45 seconds @ 48°C
 * 4) 1 minute @ 68°C
 * 5) Repeat steps 2-4 25 times
 * 6) 7 minutes @ 68°C

Electrophoresis/ Gel Procedure

Run on a 2% agarose gel.

Each lane of sample contained:
 * 10µL PCR product
 * 2µL EZ-Vision (6x)

Each lane of 100BP ladder contained:
 * 5µL ladder
 * 1µL EZ-Vision (6x)

Once lanes were loaded, the gel was run for 13 minutes at 300V.

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