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Project:Blood typing: Difference between revisions
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* Doubling quantity of template doesn't help, actually made band weaker. This indicates either that too much template hurts the reaction, or that template contains inhibitors - so doubling template doubles inhibitors. | * Doubling quantity of template doesn't help, actually made band weaker. This indicates either that too much template hurts the reaction, or that template contains inhibitors - so doubling template doubles inhibitors. | ||
* gDNA band was weaker than last time. Probably degradation. | * gDNA band was weaker than last time. Probably degradation. | ||
==Extraction and PCR (Will, Jim, Mike) 10/1/13== | |||
* Will Jim and Mike extracted DNA from their cheeks using [http://biology.clc.uc.edu/fankhauser/Labs/Genetics/Buccal_DNA_isolation/buccal_dna_images_index.html this] protocol. (15 secs swishing) | |||
* Jim and Mike's pellet was much smaller than Will's | |||
* Ran PCR with PB2 primers on these 3 + a positive control. | |||
* 25ul total PCR reaction volume - 5ul template (1ul for PC), 2.5ul each forward & reverse primers, 12.5ul SYBR green 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:Gel_10jan2013.JPG|left|400px|thumb|none|Gel 10 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: Jim PCR product (No band) | |||
*3: Mike PCR product (No band) | |||
*4: Will PCR product (No band) | |||
*5: PC PCR (band) | |||
*6: Jim gDNA (No band) | |||
*7: Mike gDNA (No band) | |||
*8: Will gDNA (Faint band) | |||
<br style="clear: both" /> | |||
'''Conclusions:''' | |||
* No or not enough DNA extracted. Possibly fault of 'whirly' technique. | |||
