Melbourne/Diagnostic Digest
From 2007.igem.org
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=====Primary & secondary Reagents Required including controls===== | =====Primary & secondary Reagents Required including controls===== | ||
*[[Melbourne/primary Restriction enzymes|Restriction enzymes and buffer]] | *[[Melbourne/primary Restriction enzymes|Restriction enzymes and buffer]] | ||
| - | *BSA | + | *[[Melbourne/primary BSA|BSA]] |
*DNA for digestion | *DNA for digestion | ||
*[[Melbourne/primary milliq|milliQ water]] | *[[Melbourne/primary milliq|milliQ water]] | ||
| Line 25: | Line 25: | ||
#**2uL appropriate 10x buffer (see table on fridge) | #**2uL appropriate 10x buffer (see table on fridge) | ||
#**2uL 10x BSA | #**2uL 10x BSA | ||
| - | #**10uL | + | #**10uL [[Melbourne/primary milliq|milliQ water]] |
#**5uL DNA | #**5uL DNA | ||
#***If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA. | #***If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA. | ||
#***Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume. | #***Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume. | ||
#Incubate for 1-3 hrs at 37 degrees. | #Incubate for 1-3 hrs at 37 degrees. | ||
| - | #Stop reaction with addition of 5uL 6x | + | #Stop reaction with addition of 5uL 6x [[Melbourne/primary dna loading| DNA Loading dye]]. |
#Can be stored at -20 or run on gel immediately. | #Can be stored at -20 or run on gel immediately. | ||
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*Ice box and [[Melbourne/primary ice|ice]] | *Ice box and [[Melbourne/primary ice|ice]] | ||
*37 degree incubator | *37 degree incubator | ||
| - | *Pipettes and [[Melbourne/primary tips|Tips | + | *Pipettes and [[Melbourne/primary tips|Tips]] |
=====References===== | =====References===== | ||
Latest revision as of 11:13, 29 September 2007
<Return to list of protocols> <Team home page>
- Applications:
- Identification of insert presence
- Time to complete protocol:
- Lab time: 15min, 15min.
- Waiting time: 1-3hours
- Approximate cost of materials: $0.00
Method from primary and secondary reagents
Primary & secondary Reagents Required including controls
- Restriction enzymes and buffer
- BSA
- DNA for digestion
- milliQ water
- Loading dye for DNA gel
Method including controls
For 20uL reation volume
- Make the following reaction mixture on ice.
- Reaction Mixture
- 0.5uL Enzyme 1
- 0.5uL Enzyme 2
- Add enzymes last only take out of the freezer once ready to add.
- 2uL appropriate 10x buffer (see table on fridge)
- 2uL 10x BSA
- 10uL milliQ water
- 5uL DNA
- If performing the same digest for multiple DNA samples make up mastermix of buffer enzyme soultion and make 15uL aliquots to which to add the DNA.
- Volume DNA may vary depending on concentration, vary water volume to achieve 20uL reaction volume.
- Reaction Mixture
- Incubate for 1-3 hrs at 37 degrees.
- Stop reaction with addition of 5uL 6x DNA Loading dye.
- Can be stored at -20 or run on gel immediately.
Equipement Required
- microcentrifuge: 1.7ml
- Ice box and ice
- 37 degree incubator
- Pipettes and Tips
References
