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
|
| 
Project name
Main project page
