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

Digest vector and PCR product

Gel Image

Things we need

• Medium latex gloves
• timers
• calculators