User:Daniel Goodman/Notebook/Weiss/2010/01/14: Difference between revisions
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==Entry title== | ==Entry title== | ||
Will Run PhyB917, PhyB, and PIF3 on a Gel | |||
[[PhyB917]] | |||
Cut with BstBI - two bands, 3579 and 5314 | |||
incubate at 65 degrees with NEBuffer 4 | |||
Nanodrop on PhyB917 DNA from 7 samples: | |||
1 380,1.88 | |||
2 441, 1.87 | |||
3 505, 1.87 | |||
4 546 1.85 | |||
5 440 1.88 | |||
6 387 1.85 | |||
7 474 1.86 | |||
Taking average, 400 ng/ul | |||
Calculate digestion mix for 7.25 aliquots: | |||
One enzyme digest | |||
calculation based on mass of DNA to be digested | |||
Mass of DNA / ug 0.2 | |||
Units of enzyme 0.2 | |||
Concentration of DNA ng/ul 400 | |||
Concentration of enzyme Units/ul 20 | |||
Overdigestion factor 2 | |||
Volume of DNA / ul 0.5 | |||
Volume of enzyme / ul 0.02 | |||
Calculate Total Volume 0.5 | |||
Actual Total Volume 15 | |||
NEB Buffer 1.5 | |||
Water 12.98 | |||
BSA 0 | |||
Water 12.98 | |||
ENZYME 0.145 | |||
BUFFER(4) 10.875 | |||
WATER 94.105 | |||
BSA 0 (none req'd for BstBII) | |||
TOAL VOL PER ALIQ: 14.5 | |||
(add 0.5 of dna to each) | |||
---- | |||
[[PIF3 (possibly)]] | |||
Cut with AvrII - two bands, 2164 and 4791 | |||
incubate at 37 deg w/ N EBuffer 4 | |||
Nanodrop on PIF3 DNA sample: | |||
357.4 ng/ul | |||
One enzyme digest | |||
calculation based on mass of DNA to be digested | |||
Mass of DNA / ug 0.2 | |||
Units of enzyme 0.2 | |||
Concentration of DNA ng/ul 350 | |||
Concentration of enzyme Units/ul 4 | |||
Overdigestion factor 2 | |||
Volume of DNA / ul 0.571428571 | |||
Volume of enzyme / ul 0.1 | |||
Calculate Total Volume 1 | |||
Actual Total Volume 15 | |||
NEB Buffer 1.5 | |||
Water 12.82857143 | |||
BSA 0 | |||
Water 12.82857143 | |||
AVRII | |||
Volume of DNA|0.571428571 | |||
Volume of enzyme|0.1 | |||
NEB Buffer|1.5 | |||
Water|12.82857143 | |||
BSA|0 | |||
Total Volume|15 | |||
Revision as of 08:35, 14 January 2010
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Entry titleWill Run PhyB917, PhyB, and PIF3 on a Gel PhyB917 Cut with BstBI - two bands, 3579 and 5314 incubate at 65 degrees with NEBuffer 4 Nanodrop on PhyB917 DNA from 7 samples: 1 380,1.88 2 441, 1.87 3 505, 1.87 4 546 1.85 5 440 1.88 6 387 1.85 7 474 1.86
Calculate digestion mix for 7.25 aliquots: One enzyme digest calculation based on mass of DNA to be digested Mass of DNA / ug 0.2 Units of enzyme 0.2 Concentration of DNA ng/ul 400 Concentration of enzyme Units/ul 20 Overdigestion factor 2 Volume of DNA / ul 0.5 Volume of enzyme / ul 0.02 Calculate Total Volume 0.5 Actual Total Volume 15 NEB Buffer 1.5 Water 12.98 BSA 0 Water 12.98 ENZYME 0.145 BUFFER(4) 10.875 WATER 94.105 BSA 0 (none req'd for BstBII) TOAL VOL PER ALIQ: 14.5 (add 0.5 of dna to each) PIF3 (possibly) Cut with AvrII - two bands, 2164 and 4791 incubate at 37 deg w/ N EBuffer 4 Nanodrop on PIF3 DNA sample: 357.4 ng/ul One enzyme digest calculation based on mass of DNA to be digested Mass of DNA / ug 0.2 Units of enzyme 0.2 Concentration of DNA ng/ul 350 Concentration of enzyme Units/ul 4 Overdigestion factor 2 Volume of DNA / ul 0.571428571 Volume of enzyme / ul 0.1 Calculate Total Volume 1 Actual Total Volume 15 NEB Buffer 1.5 Water 12.82857143 BSA 0 Water 12.82857143 AVRII Volume of DNA|0.571428571 Volume of enzyme|0.1 NEB Buffer|1.5 Water|12.82857143 BSA|0 Total Volume|15
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