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Indiana/7 July 2009
From 2009.igem.org
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
Digestion of modified pCB302
We modified pCB302 to remove Xba1 and Spe1 sites that would conflict with the iGEM multiple cloning site. To ensure that these sites were removed, we digested the plasmid and ran the digestion on a gel.
10 uL plasmid
2 uL NEB Buffer 2
.3 uL Spe1
.3 uL Hind III
7.4 uL ddH2O
We tested 16 samples. Due to the large number of samples, a master mix was made.
Running Digestion on Gel
Made .75% gel
0.75 g agarose
100 mL TBE buffer
microwave until dissolved
add 2 uL eTBr
pour into gel box
This gel revealed that an extremely small amount of plasmid was extracted using the crude method. Because of the small amount of DNA, the gel was inconclusive.
