Daniel:Notebook/HiResChrPaint/2014-1-14: Difference between revisions
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*Human Cot-1 DNA, 1 ug | *Human Cot-1 DNA, 1 ug | ||
0. Vaccuum centrifuge DNA; resuspend in | 0. Vaccuum centrifuge DNA; resuspend in 6 uL nfH2O +3 uL sodium bicarbonate buffer | ||
1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature | 1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature | ||
2. Add | 2. Add 3 uL of sodium bicarbonate buffer | ||
3 | 3. Dissolve 1 vial of reactive dye in 4 uL solvent; vortex ≥10 seconds | ||
*dye cannot be saved for later use. Use immediately! | *dye cannot be saved for later use. Use immediately! | ||
4. Add 1 uL dye to each sample | |||
5. Incubate in the dark for 1 hour | |||
6. Add 20 uL nfH20 to sample; | |||
7. Take 5 uL from sample and dilute with 45 uL nfH2O; Measure concentration in Nanodrop | |||
8. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]] | 8. [[Daniel:Protocols/CentriSep|Centri-Sep column purification]] | ||
9. Measure concentration in Nanodrop | 9. Measure concentration in Nanodrop | ||
===Labeling Results=== | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
Line 109: | Line 111: | ||
| align="center" | 1.7 | | align="center" | 1.7 | ||
| align="center" | 0.2 | | align="center" | 0.2 | ||
| align="center" | | | align="center" | 4.3 | ||
| align="center" | 15.6 | | align="center" | 15.6 | ||
| align="center" | 1.3 | | align="center" | 1.3 | ||
| align="center" | 0.3 | | align="center" | 0.3 | ||
| align="center" | 8 | | align="center" | 2.8 | ||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
Line 120: | Line 122: | ||
| align="center" | 0.6 | | align="center" | 0.6 | ||
| align="center" | 0.8 | | align="center" | 0.8 | ||
| align="center" | 0 | | align="center" | 1.0 | ||
| align="center" | 24.4 | | align="center" | 24.4 | ||
| align="center" | 0.3 | | align="center" | 0.3 | ||
| align="center" | 0.9 | | align="center" | 0.9 | ||
| align="center" | 0. | | align="center" | 0.4 | ||
|} | |} | ||
This is a big table, but there are a couple of messages to take away from this. | |||
*Excess dye causes a false DNA reading. It doesn't appear to have an effect when dye concentration is low, but when the dye concentration was high (pre-spin column), even the blank sample measured a considerable amount of DNA. I'd recommend always using a blank column and subtracting the background from future samples if pre-purification measurements are needed. | |||
*The Cot1 DNA is reproducibly eluting with dye. Given the other labeling results from previous days ([[Daniel:Notebook/HiResChrPaint/2014-1-13|1/13/2014]] and [[Daniel:Notebook/HiResChrPaint/2014-1-10|1/10/2014]]), I'd say that the Cot1 DNA, although having no amine groups, might be being labeled anyway. Or, at least, it's clogging up the column and allowing the dye to co-elute. |
Latest revision as of 21:45, 15 January 2014
Alu Control (Started 1/08/2014)[edit]
Dye Coupling Test (with Positive Control)[edit]
Repeating the dye coupling test one last time using a positive control as well.
- Following ARES Protocol
Samples
0. Vaccuum centrifuge DNA; resuspend in 6 uL nfH2O +3 uL sodium bicarbonate buffer 1. Warm DMSO and bicarbonate labeling buffer and nuclease free water to room temperature 2. Add 3 uL of sodium bicarbonate buffer 3. Dissolve 1 vial of reactive dye in 4 uL solvent; vortex ≥10 seconds *dye cannot be saved for later use. Use immediately! 4. Add 1 uL dye to each sample 5. Incubate in the dark for 1 hour 6. Add 20 uL nfH20 to sample; 7. Take 5 uL from sample and dilute with 45 uL nfH2O; Measure concentration in Nanodrop 8. Centri-Sep column purification 9. Measure concentration in Nanodrop
Labeling Results[edit]
Sample | ng/uL dsDNA | pmol/uL dye | pmol/uL ds probe | dye/100bp | ng/uL ssDNA | pmol/uL dye | pmol/uL ss probe | dye/100bp |
Alu2D-Pre Column | 170 | 115 | 113.2 | 116.6 | ||||
Blank-Pre Column | 157.4 | 111 | 103.3 | 110.6 | ||||
Cot1-Pre Column | 150.9 | 105 | 102.1 | 106.4 | ||||
V6S3-Pre Column | 160.3 | 109.2 | 109.6 | 112.9 | ||||
Alu2D-Post Column | 24.4 | 0.5 | 0.4 | 1.4 | 12.6 | 0.5 | 0.4 | 1.3 |
Blank-Post Column | 1 | 0 | 0.0 | 0.0 | 0.2 | 0 | 0.0 | 0.0 |
Cot1-Post Column | 25.9 | 1.7 | 0.2 | 4.3 | 15.6 | 1.3 | 0.3 | 2.8 |
V6S3-Post Column | 41.3 | 0.6 | 0.8 | 1.0 | 24.4 | 0.3 | 0.9 | 0.4 |
This is a big table, but there are a couple of messages to take away from this.
- Excess dye causes a false DNA reading. It doesn't appear to have an effect when dye concentration is low, but when the dye concentration was high (pre-spin column), even the blank sample measured a considerable amount of DNA. I'd recommend always using a blank column and subtracting the background from future samples if pre-purification measurements are needed.
- The Cot1 DNA is reproducibly eluting with dye. Given the other labeling results from previous days (1/13/2014 and 1/10/2014), I'd say that the Cot1 DNA, although having no amine groups, might be being labeled anyway. Or, at least, it's clogging up the column and allowing the dye to co-elute.