Yesterday I completed some re-do PCRs of Symbiodinium cp23S from the branching Porites samples Sanoosh worked on over the past summer. Some of the samples did not amplify at all, so I reattempted PCR of these samples (107, 108, 112, 116). Sample 105 amplified last summer but the sequence was lousy, so I redid that one too. After the first PCR, I obtained 1 ul of the product and diluted it 1:100 in water. I then used 1 ul of this diluted product as the template for a second round of PCR. PCR conditions were the same Sanoosh and I used last summer (based on Santos et al. 2002):
|5X Green Buffer||2.5|
|MgCl2 25 mM||2.5|
|dNTP mix 10 mM||0.6|
|Go Taq 5U/µl||0.2|
|primer 23S1 10 µM||0.5|
|primer 23S2 10 µM||0.5|
|Master Mix volume||24|
Initial denaturing period of 1 min at 95 °C, 35 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 1 min, and a final extension period of 7 min.
Samples were then run on a 1% agarose gel for 30 min at 135 volts.
Surprisingly, the first round of PCR amplified samples 107 and 112 (note: two subsamples of each were run; one that was the original extraction diluted (d) and another that was the original cleaned with Zymo OneStep PCR Inhibitor Removal kit (c)). The cleaned samples were the ones that amplified. I believe Sanoosh had tried these cleaned samples with no success.
The second round of PCR produced faint bands for both of the 108 samples. Sample 116 still did not amplify.
I cleaned the samples with the NEB Monarch Kit and shipped them today to Sequetech. I combined the two 108 samples to ensure enough DNA for sequencing.