IGEM:MIT/2007/Notebook/2007-6-12: Difference between revisions
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#Record the content of DNA in the lower right hand corner in ng/µl | #Record the content of DNA in the lower right hand corner in ng/µl | ||
==PCR amplification of EGFP== | ==PCR amplification of EGFP== | ||
# Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control | |||
# Set Reaction Tubes/Plates on Ice | |||
# 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 | |||
# Cap reaction vessel and load into a thermal cycler at 94°C | |||
# Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min | |||
# Perform the following PCR amplification | |||
## 94°C for 4 minutes | |||
## 94°C for 1 minute | |||
## 55°C for 1 minute | |||
## 72°C for 1 minute | |||
## repeat steps 2-4 35 times | |||
## 72°C for 10 minutes | |||
## 4°C forever | |||
*Run agarose gel of PCR products | *Run agarose gel of PCR products | ||
*PCR purify | *PCR purify |
Revision as of 11:34, 13 June 2007
Grad Advisors
- Morning: HD
- Afternoon: Eric
LAB WORK
- Mini-prep plasmid DNA (EGFP and NNX)
Nanodrop (~20ng/ul) Protocol
- Start ND-1000 program
- Select "nucleic acid"
- 2 µL water
- Push OK
- Wipe top and bottom
- 2 µL EB buffer (or water if used for elution)
- Select "blank"
- Wipe top and bottom
- 2 µl sample
- Select "measure"
- Record the content of DNA in the lower right hand corner in ng/µl
PCR amplification of EGFP
- Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
- Set Reaction Tubes/Plates on Ice
- 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
- Cap reaction vessel and load into a thermal cycler at 94°C
- Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
- Perform the following PCR amplification
- 94°C for 4 minutes
- 94°C for 1 minute
- 55°C for 1 minute
- 72°C for 1 minute
- repeat steps 2-4 35 times
- 72°C for 10 minutes
- 4°C forever
- Run agarose gel of PCR products
- PCR purify
- Digest vector and PCR product
Things we need
- Medium latex gloves
- timers
- calculators