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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.<br> Then using NanoDrop, I was able to quantized the DNA sample. The results are as follow: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''260/280''' | align="center" style="background:#f0f0f0;"|'''260/230''' | align="center" style="background:#f0f0f0;"|'''ng/uL''' | align="center" style="background:#f0f0f0;"|'''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 |- | |} <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.</b> <br> Add 8uL of H2O to the amount of sequencing DNA samples listed above, and then add 4uL of primer.<br> The primer location is listed below: {| {{table}} | align="center" style="background:#f0f0f0;"|'''sample''' | align="center" style="background:#f0f0f0;"|'''Forward and Reverse Primer location''' | align="center" style="background:#f0f0f0;"|'''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.<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>
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