Alice:LabNotes/2007-12-3: Difference between revisions
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<b>calculate the amount needed for sequencing: 8uL of DNA and 4 uL of primer is needed.<br> | <b>calculate the amount needed for sequencing: 8uL of DNA and 4 uL of primer is needed.<br> | ||
So use 8uL to divide the amount of ng/uL obtained. For example, when it is 10.5ng/uL, then 8ng / (10.5ng/uL) =0.8 uL. Therefore 0.8 uL of purified sample should be added. | So use 8uL to divide the amount of ng/uL obtained. For example, when it is 10.5ng/uL, then 8ng / (10.5ng/uL) =0.8 uL. Therefore 0.8 uL of purified sample should be added.</b> | ||
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Add 8uL of H2O to the amount of sequencing DNA samples listed above, and then add 4uL of primer.<br> | Add 8uL of H2O to the amount of sequencing DNA samples listed above, and then add 4uL of primer.<br> | ||
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And the above primer should be diluted on a 1:100 ratio. For rs1264899, only the sample#3 is needed for sequencing, which is the template | And the above primer should be diluted on a 1:100 ratio. For rs1264899, only the sample#3 is needed for sequencing, which is the template GM20432 cDNA. Both forward and reverse primer is used for sequencing, and they are diluted on a 2:100 ratio.<br> | ||
<b>For the submitted sequencing tubes, tube 1-5 is the sample from well#2,3,12,14,16 respectively. And tube #6 and #7 is for rs1264899 sample #3F and #3R.</b> |
Latest revision as of 21:42, 3 December 2007
I did PCR purification using column (QIAquick PCR purification) on two sets of samples, one of them is the 5 samples from expt #1(well#2,3,12,14,16), and the other one is from the new primer rs1264899.
Then using NanoDrop, I was able to quantized the DNA sample. The results are as follow:
Sample | 260/280 | 260/230 | ng/uL | sequencing amount (uL) |
well #2 | 1.67 | 2.02 | 10.5 | 0.8 |
well #3 | 1.75 | 1.31 | 26.6 | 0.3 |
well #12 | 1.67 | 1.43 | 9.6 | 0.8 |
well #14 | 1.76 | 1.68 | 12.1 | 0.8 |
well #16 | 1.71 | 1.82 | 16.4 | 0.5 |
1 | 1.71 | 1.87 | 21.0 | 0.4 |
2 | 1.79 | 1.69 | 18.9 | 0.4 |
3 | 1.64 | 1.59 | 8.4 | 0.8 |
4 | 1.79 | 1.63 | 16.7 | 0.5 |
calculate the amount needed for sequencing: 8uL of DNA and 4 uL of primer is needed.
So use 8uL to divide the amount of ng/uL obtained. For example, when it is 10.5ng/uL, then 8ng / (10.5ng/uL) =0.8 uL. Therefore 0.8 uL of purified sample should be added.
Add 8uL of H2O to the amount of sequencing DNA samples listed above, and then add 4uL of primer.
The primer location is listed below:
sample | Forward and Reverse Primer location | sequencing primer location |
well #1 | A1/A12 | B11 |
well #2 | A1/A12 | B11 |
well #3 | A2/B1 | B12 |
well #12 | A9/B8 | C7 |
well #14 | F1/F4 | F7 |
well #16 | F1/F4 | F7 |
And the above primer should be diluted on a 1:100 ratio. For rs1264899, only the sample#3 is needed for sequencing, which is the template GM20432 cDNA. Both forward and reverse primer is used for sequencing, and they are diluted on a 2:100 ratio.
For the submitted sequencing tubes, tube 1-5 is the sample from well#2,3,12,14,16 respectively. And tube #6 and #7 is for rs1264899 sample #3F and #3R.