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Project:Blood typing: Difference between revisions
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→Extraction and PCR (Will, Simon, Lui) 12/2/13
Mycoplasma (talk | contribs) |
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* 25ul total PCR reaction volume - 5ul template (1ul for PC), 2.5ul each forward & reverse primers, 12.5ul 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 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:gel12feb2013. | [[File:gel12feb2013.jpg|left|400px|thumb|none|Gel 12 Feb]] | ||
* 15ul of samples loaded, 3ul loading buffer, 7ul of ladder loaded. | * 15ul of samples loaded, 3ul loading buffer, 7ul of ladder loaded. | ||
'''From left (after 40 mins at 80V:)''' | '''From left (after 40 mins at 80V:)''' | ||
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*3: Pip PCR product (nothing) | *3: Pip PCR product (nothing) | ||
*4: A PCR product (band) | *4: A PCR product (band) | ||
*5: J PCR product (faint band) | *5: J PCR product (very faint band) | ||
*6: PC PCR product (band) | *6: PC PCR product (band) | ||
*7: Lui gDNA (nothing) | *7: Lui gDNA (nothing) | ||
*8: Pip gDNA (band) | *8: Pip gDNA (strong band) | ||
*9: A gDNA (faint band) | *9: A gDNA (faint band) | ||
*10: J gDNA (band) | *10: J gDNA (band) | ||
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'''Conclusions:''' | '''Conclusions:''' | ||
* 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. | * 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. | ||
