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Indiana/7 July 2009
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(New page: '''Miniprep cultures containing pulled parts and modified PCB302''' '''Crude Prep Method''' ''used for PCB302 preparation due to the large volume of samples''' 1) spin down 1.5 mL of o/...)
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Revision as of 19:18, 19 July 2009
Miniprep cultures containing pulled parts and modified PCB302
Crude Prep Method
used for PCB302 preparation due to the large volume of samples'
1) spin down 1.5 mL of o/n culture
2) resuspend pellet in 250 uL P1 buffer
3) add 250 uL P2 (lysis) buffer, mix by inverting
4) add 350 uL N3 buffer, mix by inverting
5) spin on max for 10 minutes
6) transfer supernatant (liquid) to clean tube
7) add equal volume of isopropanol to precipitate DNA
8) spin on max for 5 minutes
9) pour off supernatant
10) air dry tube
11) add 50 uL of TE to resuspend DNA
note: This method did not produce the desired yield. The remaining 1.5 mL of o/n culture was prepped using a filter column, as described below.
Miniprep Using a Kit
1) spin down 1.5 mL of o/n culture
2) resuspend pellet in 250 uL P1 buffer
3) add 250 uL P2 (lysis) buffer, mix by inverting
4) add 350 uL N3 buffer, mix by inverting
5) spin on max for 10 minutes
6) transfer supernatant (liquid) to a filter column
7) spin supernatant through column and discard
8) add 500 uL PB buffer, spin for 1 minute, discard liquid
9) add 500 uL PE buffer, spin 1 minute, discard liquid
10) repeat step 9
11) spin 1 minute to remove any residual PE buffer
12) transfer filter column to clean tube
13) add 30 uL EB, let sit for 1-2 minutes, then spin for 1 minute, keep liquid
14) add 20 uL EB, let sit 1-2 minutes, then spin for 1 minute, keep liquid
15) rotate filter columns 180 degrees, then spin for 30 seconds
16) transfer liquid to tube for storage
