User:Daniel Goodman/Notebook/Weiss/2010/01/14: Difference between revisions

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!Nanodrop on PhyB917 DNA from 7 samples:
!Nanodrop on PhyB917 DNA from 7 samples:
|-
|-
|1|380|1.88
|1  
|380  
|1.88
|-
|-
|2|441|1.87
|2
|441  
|1.87
|-
|-
|3|505|1.87
|3
|505
|1.87
|-
|-
|4|546|1.85
|4
|546
|1.85
|-
|-
|5|440|1.88
|5
|440
|1.88
|-
|-
|6|387|1.85
|6
|387
|1.85
|-
|-
|7|474|1.86
|7|474|1.86
Line 38: Line 50:
Calculate digestion mix for 7.25 aliquots:
Calculate digestion mix for 7.25 aliquots:


One enzyme digest
{| class="wikitable" border="1"
calculation based on mass of DNA to be digested
|-
Mass of DNA / ug 0.2
!One enzyme digest
Units of enzyme 0.2
|-
Concentration of DNA ng/ul 400
calculation based on mass of DNA to be digested||
Concentration of enzyme Units/ul 20
|-
Overdigestion factor 2
|Mass of DNA / ug
Volume of DNA / ul 0.5
|0.2
Volume of enzyme / ul 0.02
|-
Calculate Total Volume 0.5
|Units of enzyme
Actual Total Volume 15
|0.2
NEB Buffer 1.5
|-
Water 12.98
 
BSA 0
Concentration of DNA ng/ul||400
Water 12.98
|-
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
ENZYME 0.145
|-
BUFFER(4) 10.875
BUFFER(4) 10.875
|-
WATER 94.105
WATER 94.105
|-
BSA 0 (none req'd for BstBII)
BSA 0 (none req'd for BstBII)
|-
TOAL VOL PER ALIQ: 14.5
TOAL VOL PER ALIQ: 14.5
|-
(add 0.5 of dna to each)
(add 0.5 of dna to each)


Line 66: Line 102:
Cut with AvrII - two bands, 2164 and 4791
Cut with AvrII - two bands, 2164 and 4791


incubate at 37 deg w/ N EBuffer 4
incubate at 37 deg w/ N||||EBuffer 4


Nanodrop on PIF3 DNA sample:
Nanodrop on PIF3 DNA sample:
357.4 ng/ul
357.4 ng/ul


One enzyme digest
One enzyme digest||
calculation based on mass of DNA to be digested
calculation based on mass of DNA to be digested||
Mass of DNA / ug 0.2
Mass of DNA / ug||0.2
Units of enzyme 0.2
Units of enzyme||0.2
Concentration of DNA ng/ul 350
Concentration of DNA ng/ul||350
Concentration of enzyme Units/ul 4
Concentration of enzyme Units/ul||4
Overdigestion factor 2
Overdigestion factor||2
Volume of DNA / ul 0.571428571
Volume of DNA / ul||0.571428571
Volume of enzyme / ul 0.1
Volume of enzyme / ul||0.1
Calculate Total Volume 1
Calculate Total Volume||1
Actual Total Volume 15
Actual Total Volume||15
NEB Buffer 1.5
NEB Buffer||1.5
Water 12.82857143
Water||12.82857143
BSA 0
BSA||0
Water 12.82857143
Water||12.82857143


{| class="wikitable" border="1"
{| class="wikitable" border="1"
|-
|-||
!AVRII
!AVRII
|-
|-

Revision as of 09:12, 14 January 2010

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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
474|1.86

Taking average, 400 ng/ul

Calculate digestion mix for 7.25 aliquots:

calculation based on mass of DNA to be digested|| 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

One enzyme digest
Mass of DNA / ug 0.2
Units of enzyme 0.2
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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