Sam:LabNotes/Human/2009-5-22
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2nd MDA amplicons purification using MICROCON kit (#YM-100, Millipore)
Objective
- In order to estimate the DNA concentration of 2nd MDA amplicons. 20uL of 2nd MDA amplicon were purified using MICROCON kit and then measured dsDNA concentration using Nanodrop.
- It is suggesed to purify the MDA amplicon before nanodrop measuring. Otherwise the primer dimmer and salts in MDA amplicon might interfere the real dsDNA concentration.
- Only 20uL 2nd MDA amplicon was purified and measured. Since the purification of whole 2nd MAD amplicon (200uL) is not effective and time consuming.
Samples
- 200uL of 2nd MDA amplicons from:
- 2ndMDA-L4N9(8hr), 2ndMDA-L4N9(4hr), 2ndMDA-L4N8(6hr), 2ndMDA-L4N8(4hr), 2ndMDA-L4N7(6hr), 2ndMDA-L4N7(4hr), 2ndMDA-N6(6hr) and 2ndMDA-N6(4hr)
Purification
- Assemble the filter
- Transfer all DNA (about 20 uL)into a 0.5 mL tube
- Add water up to 200 uL of total volume
- Mix by p200 pipettor and transfer the mixture on the filter reservoir.
- Cap the tube and centrifuge at 14,000xg for 12 min.
- Add 20uL H2O on the filter. Invert and transfer the filter on a fresh collection tube. Centrifuge at 1000 xg for 3 min.
set up PCR reaction
- Enzyme: Qiagen Multiplex PCR kit
- Primer: Primer mix "Y"
- Template
- Row A: Un-purified: A1, D7(1/15), D7(1/30), B10(1/15), pos (29 ng gDNA), H2O
- Row B: purified: A1, D7(1/15), D7(1/30), B10(1/15), pos (29 ng gDNA)
Reaction volume test: 10uL x 13 --------- H2O 2 26 Q-sol 1 13 Enzyme 5 65
Results
File:ZhangLab 2 2009-03-21 10hr 42min-annotate.jpg
Discussion
- The kit purification protocol is working in this experiment. However, the effect of purification can't be confirmed through these results. More tests on p-pos samples may need to be done.
- A1 - the MDA amplicon from gDNA tamplate covers 4 bands(genes) which can almost serves as a positive control.