927
edits
Project:Blood typing: Difference between revisions
From London Hackspace Wiki
→PCR and Restriction digest (Tom, Will) 27/2/13
m (+cat) |
Mycoplasma (talk | contribs) |
||
| Line 677: | Line 677: | ||
* Did two reactions for each gDNA sample. 25ul total PCR reaction volume - 5ul dH20, 10ul template, 2.5ul each forward & reverse primers, 12.5ul Taq readymix | * Did two reactions for each gDNA sample. 25ul total PCR reaction volume - 5ul dH20, 10ul template, 2.5ul each forward & reverse primers, 12.5ul Taq readymix | ||
* Initial D - 96C 5 mins, then 40 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | * Initial D - 96C 5 mins, then 40 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | ||
* Incubated | * Incubated one of each PCR product with Kpn1 at 37C for 90 mins. Restriction digest mix contained 25ul PCR product, 1ul Kpn1, 3ul buffer, 3ul BSA. | ||
* Bene's samples: neither PCR product nor digest product showed, indicating PCR failed. | |||
* JJ's samples: PCR product showed up, digest product didn't, indicating either digested product was too little to see, or something destroyed the DNA during digestion, or there was inconsistency between JJ's two PCR products | |||
'''Notes/Conclusions:''' | '''Notes/Conclusions:''' | ||
* If we assume JJ's digested product was too little to see, we need to work on a better imaging system, or get more concentrated DNA out of PCR. | |||
