IGEM:MIT/2007/Notebook/2007-6-12

Grad Advisors

• Morning: HD
• Afternoon: Eric

LAB WORK

Nanodrop (~20ng/ul) Protocol

1. Start ND-1000 program
2. Select "nucleic acid"
3. 2 µL water
4. Push OK
5. Wipe top and bottom
6. 2 µL EB buffer (or water if used for elution)
7. Select "blank"
8. Wipe top and bottom
9. 2 µl sample
10. Select "measure"
11. Record the content of DNA in the lower right hand corner in ng/µl

PCR amplification of EGFP

1. Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
2. Set Reaction Tubes/Plates on Ice
3. Add the following components in a reaction vessel. Total volume should be between .5-20 µl.
   • 45 µl Platinum PCR SuperMix
   • Primers (200 nM final concentration per primer is recommended)
   • Template DNA solution
4. Cap reaction vessel and load into a thermal cycler at 94°C
5. Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
6. Perform the following PCR amplification
   1. 94°C for 4 minutes
   2. 94°C for 1 minute
   3. 55°C for 1 minute
   4. 72°C for 1 minute
   5. repeat steps 2-4 35 times
   6. 72°C for 10 minutes
   7. 4°C forever

PCR purify(microcentrifuge)

1. Harvest the bacterial cells by centrifugation at 5000rpm for 5 min. at 4˚C
2. Pipette/Decant out LB suspension
3. Resuspend pelleted bacterial cells in 0.3 ml Buffer P1 (in Falcone fridge) and transfer to a microcentrifuge tube
   • ensure that RNase A has been added to Buffer P1
4. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times (or until homogenous), and incubate at room temperature for (no more than) 5 min.
   • do not vortex
   • close Buffer P2 immediately after use
5. Add 0.4 ml of chilled Buffer N3, mix immediately and thoroughly by vigorously inverting 4-6 times, and incubate (on ice) for 5 min.
6. Centrifuge for 10min. at 13,000 rpm
7. Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting.
8. Centrifuge for 30-60 seconds on max speed. Discard flow-through
9. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.
10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl water (or Buffer EB) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Digest vector and PCR product

Figure 1: Gel Image

Things we need

• Medium latex gloves
• timers
• calculators