Brown: Team Resistance/12 June 2008

12 June 2008

Traveled to Harvard and picked up plates with several different plasmids worked on by Vivek Jain at the Mekalanos Lab

Created a culture of the pvj4 plasmid (pBAD promoter+SRRz) used in Jain Mekalanos paper • Put in incubator around 8:45pm

Miniprep Prodedure

• The purpose of this procedure is to extract plasmid DNA and separate it from chromosal DNA in a cell. All DNA (chromosal and plasmid) is denatured in the first step. Plasmid DNA, unlike chromosomal DNA, forms linked rings upon denaturation. Next, DNA is renatured with the use of buffers. Chromosomal DNA remains denatured because of the inefficiency of re-ligation of multiple pieces and strands of DNA. The plasmid DNA renatures and is thereby, separated from chromosomal DNA.
• Before starting the miniprep, the following steps should be completed with a new miniprep kit.

1. Add RNase to Buffer P1 and store in the refrigerator.
2. Add ethanol to Buffer PE.
3. Check Buffers P2 and P3 for salt precipitation and redissolve at 37 degrees C is neccessary.

• After preliminary steps have been done the miniprep is performed with the following steps:

1. 2:30 PM: Resuspend the pelleted bacterial cells in 250 microliters Buffer P1 and put in a centrifuge tube.
2. Add 250 microliters Buffer P2 and mix, invert 4-6 times. Solutions containing LyseBlue will turn blue.
3. Add 350 microliters N3 and IMMEDIATELY invert 4-6 times. Solutions with LyseBlue will turn colorless.
4. Centrifuge for 10 minutes at 13,000 rpm.
5. Apply supernatant to QIAprep spin by decanting or pipetting.
6. Centrifuge for 30-60 seconds at 13,000 rpm. Discard flow through.
7. Wash QIAprep spin column. Add .5 mL PB. Centrifuge 30-60 seconds.
8. Wash QIAprep spin column by adding .75 mL PE. Centrifuge 30-60 seconds.
9. Dsicard flow through. Centrifuge 1 minute.
10. To elute DNA, place QIAprep column in clean 1.5 mL centrifuge tube. Add 50 microliters EB or water to center of each QIAprep spin column, making sure to pipette EB right onto the cotton plug. Let stand for 1 minutes. Centrifuge for 1 minute.