L5 – L7′ (6 samples) extraxted to compare with original extracts:
L5 (9), L5′ (23), L6 (3), L6′ (17), L7 (21)
Protocol: ZR Plant/Seed DNA Kit
(Tip: weigh ‘empty’ tubes and then weigh after adding leaf tissue to calculate mass of tissue extracted)
Step 1: “vortex”
Used Fastprep (Bio101) FP 120, at speed 6 for 45s (highest time setting) and then speed 6.5 (highest speed setting) for 45s a couple of times. Machine started making a loud scraping sound, so I stopped it in the middle of the 3rd time at 6.5 and continued to the next step
Centrifuged at 10,000 rcf for 1 min
Step 3: L7 (21) <400, L5 (23) >400, L6 (17) <400, L5 (9) ~400, L6 (3) ~400, in step 3, there was nothing in the protocol that said to break the tip of the tube (under the filter) to allow the filtrate to pass, but this needed to be done before the tubes were put into the centrifuge.
Step 7: 2×50 for 2 samples, 1×50 + 1×14 for others, extra divided up.
Last step, did not mention that filtering “column” had other liquid in it, which added to the final volume of the DNA elutant.
N.B. Best gel pattern came from undiluted ‘Zymo’ DNA, so will test PCR without diluting.
