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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]] | ||
* | * 15ul of samples loaded, 6ul of ladder loaded. | ||
'''From left (after | '''From left (after 40 mins at 80V:)''' | ||
*1: 100-3000bp ladder. Ladder [http://www.nbsbio.co.uk/product.asp?pID=8951&cID=33 key]. | *1: 100-3000bp ladder. Ladder [http://www.nbsbio.co.uk/product.asp?pID=8951&cID=33 key]. | ||
*2: Simon PCR product (nothing) | *2: Simon PCR product (nothing) | ||
*3: Will PCR product (faint gDNA band) | *3: Will PCR product (faint gDNA band, not visible in photo) | ||
*4: PC PCR product (nothing) | *4: PC PCR product (nothing) | ||
*5: Simon gDNA (band) | *5: Simon gDNA (band, not visible in photo) | ||
*6: Will gDNA (band) | *6: Will gDNA (band) | ||
| Line 518: | Line 518: | ||
== PCR (Will, Simon) 14/1/13== | == PCR (Will, Simon) 14/1/13== | ||
* Repeat PCR on samples from 13/1. Samples | * Repeat PCR on samples from 13/1. Samples centrifuged 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 | * 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) | * 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. | * 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]] | [[File:gel14jan2013.JPG|left|400px|thumb|none|Gel 14 Jan]] | ||
* PCR left overnight. 15ul of samples loaded, | * PCR left overnight. 15ul of samples loaded, 10ul of ladder loaded. | ||
'''From left (after 35 mins at 80V:)''' | '''From left (after 35 mins at 80V:)''' | ||
*1: 100-3000bp ladder. Ladder [http://www.nbsbio.co.uk/product.asp?pID=8951&cID=33 key]. | *1: 100-3000bp ladder. Ladder [http://www.nbsbio.co.uk/product.asp?pID=8951&cID=33 key]. | ||
*2: Simon PCR product | *2: Simon PCR product (Nothing) | ||
*3: Will PCR product | *3: Will PCR product (Nothing) | ||
*4: PC SYBR green Taq PCR product | *4: PC SYBR green Taq PCR product (Faint band) | ||
*5 PC Taq PCR product | *5 PC Taq PCR product (Band) | ||
*6: Simon gDNA | *6: Simon gDNA (Nothing) | ||
*7: Will gDNA | *7: Will gDNA (Faint band in well + unexplained blob) | ||
<br style="clear: both" /> | <br style="clear: both" /> | ||
'''Conclusions:''' | '''Conclusions:''' | ||
* This time PCR worked on the PCs (better on straight Taq readymix than SYBR green Taq readymix). So something wrong with extractions. Either contamination or degradation (gDNA bands weak or non existent compared to same samples 24 hours earlier) | |||
