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Project:Blood typing: Difference between revisions
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* Initial D - 96C 5 mins, then 35 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | * Initial D - 96C 5 mins, then 35 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | ||
[[File:gel13jan2013.JPG|left|400px|thumb|none|Gel 13 Jan]] | [[File:gel13jan2013.JPG|left|400px|thumb|none|Gel 13 Jan]] | ||
* PCR left overnight. 15ul of samples loaded, 6ul of ladder loaded. | |||
'''From left (after 35 mins at 80V:)''' | |||
*1: 100-3000bp ladder. Ladder [http://www.nbsbio.co.uk/product.asp?pID=8951&cID=33 key]. | |||
*2: Simon PCR product (nothing) | |||
*3: Will PCR product (faint gDNA band) | |||
*4: PC PCR product (nothing) | |||
*5: Simon gDNA (band) | |||
*6: Will gDNA (band) | |||
<br style="clear: both" /> | |||
'''Conclusions:''' | |||
* Seems to have been a PCR failure rather than extraction failure. No idea why, but most likely problems with taq or primers, or pipetting error. Also the template had a yellowish tinge, which is something past PCR failures have also had. | |||
== PCR (Will, Simon) 14/1/13== | |||
* Repeat PCR on samples from 13/1. Samples centrifuges to remove debris, but yellowish tint remained. | |||
* Ran PCR with PB1 primers on these two + 1 positive control with SYBR green Taq readymix + 1 PC with Taq readymix | |||
* 25ul total PCR reaction volume - 5ul template (1ul for PC), 2.5ul each forward & reverse primers, 12.5ul SYBR green Taq readymix / Taq readymix, 2.5ul dH20 (6.5ul for PC) | |||
* Initial D - 96C 5 mins, then 35 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | |||
[[File:gel14jan2013.JPG|left|400px|thumb|none|Gel 14 Jan]] | |||
* PCR left overnight. 15ul of samples loaded, 6ul of ladder loaded. | * PCR left overnight. 15ul of samples loaded, 6ul of ladder loaded. | ||
'''From left (after 35 mins at 80V:)''' | '''From left (after 35 mins at 80V:)''' | ||
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*2: Simon PCR product | *2: Simon PCR product | ||
*3: Will PCR product | *3: Will PCR product | ||
*4: PC PCR product | *4: PC SYBR green Taq PCR product | ||
*5: Simon gDNA | *5 PC Taq PCR product | ||
* | *6: Simon gDNA | ||
*7: Will gDNA | |||
<br style="clear: both" /> | <br style="clear: both" /> | ||
'''Conclusions:''' | '''Conclusions:''' | ||
