Noi/NOTES/2014-2-28: Difference between revisions

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= MONOD test on human plasma/serum =
= MONOD test on human plasma/serum =
* '''Note from Dr. Zhang'''
* '''Note from Dr. Zhang'''
::We will get some human plasma and serum from cancer center for the MONOD project. This would be different from all the BSPP experiments we did before, as we are dealing with circulating cell-free DNA from human patients.
::'''"''' We will get some human plasma and serum from cancer center for the MONOD project. This would be different from all the BSPP experiments we did before, as we are dealing with circulating cell-free DNA from human patients.
   
   
::One major challenge is to recover enough DNA for BSPP, as the yield of DNA extraction from plasma/serum is very low in general, and seems to be quite variable depending on the DNA extraction kits (we don’t want whole blood or buffy coats as they contain mostly white blood cells).
::One major challenge is to recover enough DNA for BSPP, as the yield of DNA extraction from plasma/serum is very low in general, and seems to be quite variable depending on the DNA extraction kits (we don’t want whole blood or buffy coats as they contain mostly white blood cells).
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::Please keep track of the kits when they arrive, and write up an experimental protocol based on the manuals of these kits. The materials are precious so please confirm the plan with me before processing the samples. We will do '''picogreen quantification''' and '''Bioanalyzer analysis''' to determine the yield and size distribution (cell-free DNA are fragmented in general). We can move forward with BSPP if there is enough material.
::Please keep track of the kits when they arrive, and write up an experimental protocol based on the manuals of these kits. The materials are precious so please confirm the plan with me before processing the samples. We will do '''picogreen quantification''' and '''Bioanalyzer analysis''' to determine the yield and size distribution (cell-free DNA are fragmented in general). We can move forward with BSPP if there is enough material.
   
   
::We will received '''a set of four tubes of plasma from one individual''', and '''another four tubes of serum from another'''. Each tube has roughly '''200ul of biofluid'''.
::We will received '''a set of four tubes of plasma from one individual''', and '''another four tubes of serum from another'''. Each tube has roughly '''200ul of biofluid''' '''"'''.
{| {{table}} class = wikitable
{| {{table}} class = wikitable
| align="center" style="background:#f0f0f0;"|'''Kit'''
| align="center" style="background:#f0f0f0;"|'''Kit'''

Revision as of 16:27, 28 February 2014

MONOD test on human plasma/serum

  • Note from Dr. Zhang
" We will get some human plasma and serum from cancer center for the MONOD project. This would be different from all the BSPP experiments we did before, as we are dealing with circulating cell-free DNA from human patients.
One major challenge is to recover enough DNA for BSPP, as the yield of DNA extraction from plasma/serum is very low in general, and seems to be quite variable depending on the DNA extraction kits (we don’t want whole blood or buffy coats as they contain mostly white blood cells).
We will received a set of four tubes of plasma from one individual, and another four tubes of serum from another. Each tube has roughly 200ul of biofluid.
I’ve ordered four kits (QIAamp blood mini, Lifetech ChargeSwitch, Clontech Nucleospin, ZymoBead). Our first step is to determine the yields with these kits, and for the bead-based kits, to try direct bisulfite conversion of DNA on the beads without washing and elution.
Please keep track of the kits when they arrive, and write up an experimental protocol based on the manuals of these kits. The materials are precious so please confirm the plan with me before processing the samples. We will do picogreen quantification and Bioanalyzer analysis to determine the yield and size distribution (cell-free DNA are fragmented in general). We can move forward with BSPP if there is enough material.
We will received a set of four tubes of plasma from one individual, and another four tubes of serum from another. Each tube has roughly 200ul of biofluid ".
Kit Vendor Catalog # Protocol Note
QIAamp DNA Blood Mini Kit (50) QIAGEN 51104 QIAamp Blood Mini Kit Manual Column-based method, page 26-28
ChargeSwitch® gDNA Serum Kit, 0.2-1 mL Life Technologies CS11040 ChargeSwitch® gDNA Serum Kit Manual Bead-based method
NucleoSpin® Plasma XS Fisher Scientific/Marcherey Nagel NC9959409 NucleoSpin® Plasma XS kit manual Column-based method, Estimeted delivert date: 2014-03-14
ZymoBead™ Genomic DNA Kit Zymo Research D3004 ZymoBead™ Genomic DNA Kit manual Bead-based method

Experimental plan

  • I will confirm with Dr. Zhang about the volume of serum/plasma to use for each kit since each one uses different volume. Shall I used recommended volume of each kit and then compare the yield to normalized input?

Column-based methods

QIAamp DNA Blood Mini Kit

NucleoSpin® Plasma XS kit

Bead-based methods

ChargeSwitch® gDNA 1 ml Serum kit

  • Upon receipt, store proteinase K at 4C
Product Specifications
Starting Material:    0.2-1 ml of fresh or frozen human serum, with or without EDTA
Elution Volume:       50 μl
DNA Yield:            1-200 ng
DNA Size:             50 bp-1.5 kb

ZymoBead™ Genomic DNA kit