Daniel:Notebook/HiResChrPaint/2013-5-8

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Probe Prep Test (Started 4/22/2013)[edit]

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Column Purification vs. Ethanol Precipitation[edit]

Ethanol Precipitation (continued from 5/6/2013)[edit]

5. Centrifuged 30 min at 10000 rpm at 4C
6. Removed supernatant and added 750 uL chilled EtOH
7. Centrifuged 15 min at 10000 rpm at 4C
8. Removed supernatant and dried in hood
9. Resuspended in 10 uL nf H20

TBU Gel comparison[edit]

Protocol 2

1. Gel lanes
Sample 0.5 Ladder 1 uL Ladder Original Column (1uL) Column (2uL) EtOH (1 uL) A EtOH (2uL) A EtOH (1 uL) B EtOH (2uL) B
uL sample 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5
uL TBE 9.5 9 9.5 9.5 9.5 9.5 9.5 9.5 9.5
uL 2X buffer 10 10 10 10 10 10 10 10 10
Loaded 4 4 4 4 4 4 4 4 4
Dilution 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
2. Heat shock 10 minutes at 75 C
3. Run gel for 24 minutes at 250V
4. Stain with 3 uL SYBR gold for 20 minutes

Gel Results[edit]

Volume Report: File:PrimerQuant 050813.xls

File:PrimerQuant 050813.jpg

File:PrimerQuant 050813 Volume.jpg

Quantification Results[edit]

  ng/uL Yield Fold Increase (2uL/1uL)
Original 1926.4 139.8
1 uL column 906.6 65.8
2 uL column 1337.0 97.0 1.5
1 uL EtOH A 205.5 14.9
2 uL EtOH A 676.0 49.1 3.3
1 uL EtOH B 204.6 14.8
2 uL EtOH B 549.2 39.9 2.7

Discussion[edit]

Looks like the column purification is better than Ethanol precipitation once we get this low.  As such, I'm going to start 
my next probe set with more DNA and then when I get to the lambda digestion I'll purify with the column.  I also think that 
the amount of starting material for the ethanol precipitation affects the yield.  I think that starting with more DNA will 
give a higher % yield, because some DNA will always be left in solution.  This would be confirmed if the gel quantification 
results in less than 1/2 yields in the 1uL samples compared with the 2.