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Project:Blood typing: Difference between revisions
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* JJ and Bene extracted DNA from cheeks on wed 20th using [http://biology.clc.uc.edu/fankhauser/Labs/Genetics/Buccal_DNA_isolation/buccal_dna_images_index.html this] protocol. Small amount of cheek cells for each. | * JJ and Bene extracted DNA from cheeks on wed 20th using [http://biology.clc.uc.edu/fankhauser/Labs/Genetics/Buccal_DNA_isolation/buccal_dna_images_index.html this] protocol. Small amount of cheek cells for each. | ||
* On 23rd | * On 23rd ran PCR with PB1 primers on these + a positive control. | ||
* 50ul total PCR reaction volume - 5ul dH20 (13ul for PC), 10ul template (2ul for PC), 5ul each forward & reverse primers, 25ul Taq readymix | * 50ul total PCR reaction volume - 5ul dH20 (13ul for PC), 10ul template (2ul for PC), 5ul each forward & reverse primers, 25ul Taq readymix | ||
* Initial D - 96C 5 mins, then 40 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | * Initial D - 96C 5 mins, then 40 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | ||
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* Bene did a new extraction using same protocol. | * Bene did a new extraction using same protocol. | ||
* Ran PCR with PB1 primers on this + JJ's old sample + Will's sample from | * Ran PCR with PB1 primers on this + JJ's old sample + Will's sample from 17/2 (1 with 50ul total volume, 1 with 25ul). | ||
* 50ul total PCR reaction volume - 5ul dH20 (13ul for PC), 10ul template (2ul for PC), 5ul each forward & reverse primers, 25ul Taq readymix | * 50ul total PCR reaction volume - 5ul dH20 (13ul for PC), 10ul template (2ul for PC), 5ul each forward & reverse primers, 25ul Taq readymix. (Halve quantities for Will2) | ||
* Initial D - 96C 5 mins, then 40 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | * Initial D - 96C 5 mins, then 40 cycles of D-96C 1 min, A-55C 30 secs, E-72C 1 min. | ||
[[File: | [[File:gel2-23feb2013.JPG|left|400px|thumb|none|Gel 2 23 Feb]] | ||
* 20ul of PCR samples loaded, | * 20ul of PCR samples loaded, 3ul loading buffer. Same gel used hence lanes 4+5 same as that one. | ||
*4: JJ gDNA (band) | *4: JJ gDNA (band) | ||
*5: Bene gDNA (band) | *5: Bene gDNA (band) | ||
*6: JJ PCR | |||
*7: Bene PCR | |||
*8: Will PCR (50ul) | |||
*9: Will PCR (25ul) | |||
<br style="clear: both" /> | <br style="clear: both" /> | ||
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'''Notes/Conclusions:''' | '''Notes/Conclusions:''' | ||
* | * This seems to indicate pretty clearly that doubling the reaction quantity caused the PCRs to fail. Will try again on JJ's and Bene's with 25ul. | ||
