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/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/ 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 / 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
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