927
edits
Project:Blood typing: Difference between revisions
From London Hackspace Wiki
→Extraction and PCR (Will, Simon, Lui) 12/2/13
Mycoplasma (talk | contribs) |
|||
| Line 581: | Line 581: | ||
* Lui's extraction didn't contain enough liquid after boiling step, so he added more water afterwards and mixed. This could be responsible for extraction failure. Not sure reasons for variations in RCA extractions. | * Lui's extraction didn't contain enough liquid after boiling step, so he added more water afterwards and mixed. This could be responsible for extraction failure. Not sure reasons for variations in RCA extractions. | ||
==Extraction and PCR (David, Will) 17/2/13== | |||
* David and Will extracted DNA from cheeks using [http://biology.clc.uc.edu/fankhauser/Labs/Genetics/Buccal_DNA_isolation/buccal_dna_images_index.html this] protocol. | |||
* Ran PCR with PB1 primers on this + Victor's extraction from 13/2 + a positive control. | |||
* 25ul total PCR reaction volume - 2.5ul dH20 (6.5ul for PC), 5ul template (1ul for PC), 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. | |||
[[File:gel17feb2013.jpg|left|400px|thumb|none|Gel 17 Feb]] | |||
* 15ul of samples loaded, _ul loading buffer, _ul of ladder loaded. | |||
'''From left (after 40 mins at 80V:)''' | |||
*1: 100-3000bp ladder. Ladder [http://www.nbsbio.co.uk/product.asp?pID=8951&cID=33 key]. | |||
*2: Victor PCR product | |||
*3: David PCR product | |||
*4: Will PCR product | |||
*5: PC PCR product | |||
*6: PC gDNA | |||
*7: Victor gDNA | |||
*8: David gDNA | |||
*9: Will gDNA | |||
<br style="clear: both" /> | |||
'''Notes/Conclusions:''' | |||
* Increased PCR cycles from 35 to 40 | |||
* First pipetting of Will gDNA went over gel instead of into hole. We repeated but could explain diffusion + weak band | |||
