IGEM:MIT/2006/Notebook/2006-7-5: Difference between revisions
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#*'''.5 μL Dpn 1''' | #*'''.5 μL Dpn 1''' | ||
#Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever. | #Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever. | ||
#Did a PCR cleanup & nanodrop: concentration of digested | #Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/μL | ||
#'''run a gel before moving on to ligation !!''' | #'''run a gel before moving on to ligation !!''' | ||
==ATF1 PCR Product Xba/Pst Digest== | ==ATF1 PCR Product Xba/Pst Digest== |
Revision as of 07:47, 5 July 2006
BAMT PCR Product Eco/Pst Digest
- We are cutting the BAMT pcr product (using EcoRI and PstI)--
- Total reaction volume is 50μL
- the amount of dna desired is about 800 nanograms
- BAMT pcr product at 58.8 ng/μL -----add 13.6 μL
- we are also going to use Dpn1 only in BAMT cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
- BAMT pcr cleanup product cut reaction:
- 28.9 μL water
- 5 μL NEB buffer 2
- .5 μL BSA
- 13.6 μL BAMT pcr cleanup product (concentration = 50 ng/μL)
- .75 μL Pst1
- .75 μL EcoR1
- .5 μL Dpn 1
- Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
- Did a PCR cleanup & nanodrop: concentration of digested BAMT = ??ng/μL
- run a gel before moving on to ligation !!
ATF1 PCR Product Xba/Pst Digest
- We are cutting the ATF1 pcr product (using EcoRI and PstI)--
- Total reaction volume is 50μL
- the amount of dna desired is about 800 nanograms
- ATF1 pcr product at 30.8 ng/μL -----add 26 μL
- we are also going to use Dpn1 only in ATF1 cut reaction to destroy dna that is not a pcr product (i.e. get rid of extra template)
- ATF1 Digest:
- 16.5 μL water
- 5 μL NEB buffer 3
- .5 μL BSA
- 26 μL ATF1 pcr cleanup product (concentration = 50 ng/μL)
- .75 μL Pst1
- .75 μL XbaI
- .5 μL Dpn 1
- Incubate tube in 37c room for 1-6 hours, then 20 mins at 80deg C to heat inactivate the enzyme, then 4deg forever.
- Did a PCR cleanup & nanodrop: concentration of digested SAMT = 10.4ng/μL
- run a gel before moving on to ligation !!
Miniprep
- Did minipreps of A, C, E BSMT liquid cultures
pSB1A3-1 bkb Xba/Pst Digest
- BACKBONE cut reaction (in order added, following the Knight Lab restriction digest protocol):
- 35 μL water
- 5 μL NEB buffer 3
- .5 μL BSA
- 8 μL backbone plasmid
- .75 μL PstI
- .75 μL XbaI