Indiana/7 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

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.