User:Daniel Goodman/Notebook/Weiss/2010/01/14

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Prep for Plasmid Digestion Tomorrow

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
474|1.86

Taking average, 400 ng/ul

Calculate digestion mix for 7.25 aliquots:

calculation based on mass of DNA to be digested||
One enzyme digest
Mass of DNA / ug 0.2
Units of enzyme 0.2
Concentration of DNA ng/ul400
Concentration of enzyme Units/ul20
Overdigestion factor2
Volume of DNA / ul0.5
Volume of enzyme / ul0.02
Calculate Total Volume0.5
Actual Total Volume15
NEB Buffer1.5
Water12.98
BSA0
Water12.98
ENZYME0.145
BUFFER(4)10.875
WATER94.105
BSA0 (none req'd for BstBII)
TOAL VOL PER ALIQ14.5
(add 0.5 of dna to each)

PIF3 (possibly)

Cut with AvrII - two bands, 2164 and 4791

incubate at 37 deg w/ NEBuffer 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 / ug0.2
Units of enzyme0.2
Concentration of DNA ng/ul350
Concentration of enzyme Units/ul4
Overdigestion factor2
Volume of DNA / ul0.571428571
Volume of enzyme / ul0.1
Calculate Total Volume1
Actual Total Volume15
NEB Buffer1.5
Water12.82857143
BSA0
Water12.82857143
MADE MASTER MIX FOR BSTBI AND ADDED ENZYME, BUFFER, AVRII; add DNA and water for AVRII and run digestion tomorrow!
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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