Noi/NOTES/2014-2-28: Difference between revisions
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= MONOD test on human plasma/serum = | = MONOD test on human plasma/serum = | ||
[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar]] | |||
* '''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). | ||
Line 8: | Line 9: | ||
::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. | ::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. | ::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. | ||
Line 19: | Line 20: | ||
| align="center" style="background:#f0f0f0;"|'''Note''' | | align="center" style="background:#f0f0f0;"|'''Note''' | ||
|- | |- | ||
| QIAamp DNA Blood Mini Kit (50)||QIAGEN||51104||[[Media:QIAamp-DNAMini-and-BloodMini-Handbook.pdf|QIAamp Blood Mini Kit Manual]]||Column-based method, page 26-28 | | QIAamp DNA Blood Mini Kit (50)||QIAGEN||51104||[[Media:QIAamp-DNAMini-and-BloodMini-Handbook.pdf|QIAamp Blood Mini Kit Manual]]||Column-based method, page 26-28<br> arrived 03/04/2014 | ||
|- | |- | ||
| ChargeSwitch® gDNA Serum Kit, 0.2-1 mL||Life Technologies||CS11040||[[Media:chargeswitch_gdna_1mlserum_manual.pdf|ChargeSwitch® gDNA Serum Kit Manual]]||Bead-based method | | ChargeSwitch® gDNA Serum Kit, 0.2-1 mL||Life Technologies||CS11040||[[Media:chargeswitch_gdna_1mlserum_manual.pdf|ChargeSwitch® gDNA Serum Kit Manual]]||Bead-based method<br> arrived 03/03/2014 | ||
|- | |- | ||
| NucleoSpin® Plasma XS||Fisher Scientific/Marcherey Nagel||NC9959409 ||[[Media:UM_CircDNAPlasma_NSXS.pdf|NucleoSpin® Plasma XS kit manual]]||Column-based method, | | NucleoSpin® Plasma XS||Fisher Scientific/Marcherey Nagel||NC9959409 ||[[Media:UM_CircDNAPlasma_NSXS.pdf|NucleoSpin® Plasma XS kit manual]]||Column-based method, Estimated delivery date: 03/14/2014<br> arrived 03/06/2014 | ||
|- | |- | ||
| ZymoBead™ Genomic DNA Kit||Zymo Research||D3004||[[Media:ZymoBead-GenomicDNA-Kit.pdf|ZymoBead™ Genomic DNA Kit manual]]||Bead-based method | | ZymoBead™ Genomic DNA Kit||Zymo Research||D3004||[[Media:ZymoBead-GenomicDNA-Kit.pdf|ZymoBead™ Genomic DNA Kit manual]]||Bead-based method<br> arrived 03/04/2014 | ||
|} | |} | ||
== Experimental plan == | == Experimental plan == | ||
Line 61: | Line 62: | ||
DNA size range: 50-1000bp | DNA size range: 50-1000bp | ||
DNA yield from human plasma: DNA amounts from less than 0.1 ng DNA per mL of plasma up to several 100 ng DNA per mL of plasma have been reported | DNA yield from human plasma: DNA amounts from less than 0.1 ng DNA per mL of plasma up to several 100 ng DNA per mL of plasma have been reported | ||
: - Add 1.35mL of Proteinase Buffer (PB) to 30mg of lyophilized Proteinase K for 50 prep kit. | |||
==== Procedures ==== | ==== Procedures ==== | ||
: # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min<br> | : # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min<br> | ||
: <u>Preparing lysate</u> | : <u>Preparing lysate</u> | ||
:: - Add '''240ul''' of '''plasma/serum''' to 1.5mL tube. | :: - Add '''240ul''' of '''plasma/serum''' to 1.5mL tube. --> ''' I will add 200ul''' | ||
:: - Add '''20ul''' of '''proteinase K''' to plasma/serum sample | :: - Add '''20ul''' of '''proteinase K''' to plasma/serum sample | ||
:: - Mix and incubate at 37C for 10min | :: - Mix and incubate at 37C for 10min | ||
: <u>Binding DNA</u> | : <u>Binding DNA</u> | ||
:: - Add '''1.5 volume''' (360ul) of ''' | :: - Add '''1.5 volume''' (360ul) of '''Buffer BB''' (keep sample:binding buffer ration 1:1.5 v/v) --> '''should be 300ul of Buffer BB''' | ||
:: - Invert the tube 3x and vortex for 3s. Spin down the tube briefly. | :: - Invert the tube 3x and vortex for 3s. Spin down the tube briefly. | ||
:: - Load 600ul of the mixture to a Nucleospin Plasma XS column | :: - Load 600ul of the mixture to a Nucleospin Plasma XS column | ||
Line 82: | Line 84: | ||
:: - Centrifuge at 11,000xg for 30s | :: - Centrifuge at 11,000xg for 30s | ||
:: - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol | :: - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol | ||
== Bead-based methods == | == Bead-based methods == | ||
=== 3. ChargeSwitch® gDNA 1 ml Serum kit === | === 3. ChargeSwitch® gDNA 1 ml Serum kit === | ||
Line 142: | Line 145: | ||
=== 4. ZymoBead™ Genomic DNA kit === | === 4. ZymoBead™ Genomic DNA kit === | ||
: -This protocol is for whole blood, serum and plasma samples | : -This protocol is for whole blood, serum and plasma samples | ||
==== Procedures ==== | |||
: -Before starting, add beta-mercaptoethanol to Genomic Lysis Buffer to a final dilution of 0.5% (v/v). Vortex ZymoBead to resuspend | : -Before starting, add beta-mercaptoethanol to Genomic Lysis Buffer to a final dilution of 0.5% (v/v). Vortex ZymoBead to resuspend | ||
: <u>Preparing lysate and binding DNA</u> | : <u>Preparing lysate and binding DNA</u> | ||
Line 166: | Line 170: | ||
:: - Collect the spnt to the microcentrifuge tube. | :: - Collect the spnt to the microcentrifuge tube. | ||
=== Things to check === | === Things to check === | ||
: - Beta-mercaptoethanol | : - Beta-mercaptoethanol --> Yes | ||
: - Magnetic rack for 1.5mL tube is working well? | : - Magnetic rack for 1.5mL tube is working well? --> Yes | ||
: - Need to use carriers? | : - Need to use carriers? | ||
: - ZymoBeads allows for 50ul samples. Do I need to do multiple tubes for total 200ul sample? Double check with Dr. Zhang the total volume required to test for each kit. | : - ZymoBeads allows for 50ul samples. Do I need to do multiple tubes for total 200ul sample? Double check with Dr. Zhang the total volume required to test for each kit. | ||
: - How to combine DNA purification with bisulfite conversion for bead-based methods. | : - How to combine DNA purification with bisulfite conversion for bead-based methods. | ||
:: Recommended paper from Dr. Zhang: [[Media:Bailey_VJ_etal2010.pdf|Bailey VJ et al, 2010 ]] | :: '''Recommended paper''' from Dr. Zhang: [[Media:Bailey_VJ_etal2010.pdf|Bailey VJ et al, 2010 ]] | ||
:: - In this paper, the step of cell lysis and bisulfite conversion were performed in the same tube and used the same magnetic beads in both steps. The house made buffers of bisulfite conversion step were prepared to compatible with the magnetic beads used in this experiment. | |||
:: - [[Media:BaileyVJ_etal2010_Supp_Methods.pdf|BaileyVJ_etal2010_Supp_Methods]] | |||
==== 2014-03-04 ==== | |||
: - Received 3 case samples from Sharmeela | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Case/Accession #''' | |||
| align="center" style="background:#f0f0f0;"|'''Project ID #''' | |||
| align="center" style="background:#f0f0f0;"|'''Typr/Amount''' | |||
| align="center" style="background:#f0f0f0;"|'''Daigonosis''' | |||
|- | |||
| AA005||UCSD004.1||4-BC,P,S||Eosinophilia | |||
|- | |||
| AA007||UCSD004.2||4-BC,P,S||Anemia | |||
|- | |||
| AA008||UCSD004.3||4-BC,P, 3S||Anemia | |||
|} | |||
:: Note: SampleUCSD004.3 had only 3 aliquots of serum | |||
:: - White caps : Buffy coat (BC) | |||
:: - Green caps: Plasma (P) | |||
:: - Blue caps : Serum (S) | |||
==== 2014-03-10 ==== | |||
'''Discussion with Dr. Zhang''' | |||
=== Next things to check === | |||
: - Waiting for Van (Dr. Sah's lab manager) for plate reader demonstration | |||
: - Confirm with Dr. Zhang where can I use Bioanalyzer | |||
:: -> At Dr. Palsson's lab | |||
: - Should I extract all samples (3 cases) or try out only one sample? | |||
:: -> Extract sample #1 & #2 and save #3 | |||
: - For sample #3, which has only 3 tube of serum, what are the three kits out of four I should use for DNA extraction? | |||
:: -> No need to extract sample #3 for now | |||
* Continued on: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-3-11]] |
Latest revision as of 00:54, 30 March 2014
MONOD test on human plasma/serum[edit]
- 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 arrived 03/04/2014 |
ChargeSwitch® gDNA Serum Kit, 0.2-1 mL | Life Technologies | CS11040 | ChargeSwitch® gDNA Serum Kit Manual | Bead-based method arrived 03/03/2014 |
NucleoSpin® Plasma XS | Fisher Scientific/Marcherey Nagel | NC9959409 | NucleoSpin® Plasma XS kit manual | Column-based method, Estimated delivery date: 03/14/2014 arrived 03/06/2014 |
ZymoBead™ Genomic DNA Kit | Zymo Research | D3004 | ZymoBead™ Genomic DNA Kit manual | Bead-based method arrived 03/04/2014 |
Experimental plan[edit]
- 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 use recommended volume of each kit and then compare the yield to normalized input?
Column-based methods[edit]
1. QIAamp DNA Blood Mini Kit[edit]
- - Resuspend lyophilized QIAGEN Protease with 1.2 ml protease solvent.
- - Use carrier DNA (e.g., poly dA, poly dT, poly dA:dT) when the sample is low-copy (i.e., when <10,000 copies are present)
Starting material: 200ul for blood, plasma, serum or buffy coat
Procedures[edit]
- Preparing lysate
- - Pipet 20 µl of QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml tube
- - Add 200 µl sample to the tube. If the volume of sample less than 200ul, adjust volume with PBS
- - Add 200 µl of Buffer AL to the sample. Mix by pulse-vortexing for 15 s
- - Incubate at 56°C for 10 min
- Binding DNA
- - Briefly spin down the tube before adding 200ul 100% EtOH. Mix again by pulse-vortexing for 15 s and briefly spin down
- - Transfer spnt to QIAamp Mini spin column & centrifuge at 6,000xg for 1 min
- Washing DNA
- - Transfer QIAamp Mini spin column in a clean 2 ml collection tube
- - Add 500 µl of Buffer AW1 & centrifuge at 6,000xg for 1 min
- - Transfer QIAamp Mini spin column in a clean 2 ml collection tube
- - Add 500 µl of Buffer AW2 & centrifuge at 20,000xg for 3min
- Eluting DNA
- - Transfer the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at 20,000xg for 1 min (full speed)
- - Place the QIAamp Mini spin column in a clean 1.5 ml tube & add appropriate volume of Buffer AE or distilled water (manual recommends 200ul).
- - Incubate at RT for 5 min & centrifuge at 6,000xg for 1 min
2. NucleoSpin® Plasma XS kit[edit]
Product specifications Starting material: Up to 240 μL EDTA plasma Elution volume: 5–30 μL Average yield: 0.1–100 ng per mL plasma DNA size range: 50-1000bp DNA yield from human plasma: DNA amounts from less than 0.1 ng DNA per mL of plasma up to several 100 ng DNA per mL of plasma have been reported
- - Add 1.35mL of Proteinase Buffer (PB) to 30mg of lyophilized Proteinase K for 50 prep kit.
Procedures[edit]
- # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min
- Preparing lysate
- - Add 240ul of plasma/serum to 1.5mL tube. --> I will add 200ul
- - Add 20ul of proteinase K to plasma/serum sample
- - Mix and incubate at 37C for 10min
- Binding DNA
- - Add 1.5 volume (360ul) of Buffer BB (keep sample:binding buffer ration 1:1.5 v/v) --> should be 300ul of Buffer BB
- - Invert the tube 3x and vortex for 3s. Spin down the tube briefly.
- - Load 600ul of the mixture to a Nucleospin Plasma XS column
- - Centrifuge at 2,000xg for 30s. Centrifuge at higher speed at 11,000xg for further 5s.
- Washing DNA
- - Transfer Nucleospin column to a new collecting tube
- - Add 500ul of buffer WB onto the column. Centrifuge at 11,000xg for 30s.
- - Place column into new collecting tube
- - Add 250ul of buffer WB to the column. Centrifuge at 11,000xg for 3min
- Eluting DNA
- - Place column to 1.5mL tube
- - Add 20ul of Elution buffer to the column (range 5-30ul)
- - Centrifuge at 11,000xg for 30s
- - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol
Bead-based methods[edit]
3. ChargeSwitch® gDNA 1 ml Serum kit[edit]
- - 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
Procedures[edit]
Suggested reagent volumes for different sample volumes
Reagent\sample volume (S) 1mL 800ul 600ul 400ul 200ul A volume: ChargeSwitch Lysis Buffer (L19) 700 ul 560 ul 420 ul 280 ul 140 ul Proteinase K 30 ul 30 ul 30 ul 30 ul 30 ul Rnase A (optional) 5 ul 5 ul 5 ul 5 ul 5 ul B volume: ChargeSwitch Purification Buffer (N7) 250 ul 200 ul 150 ul 100 ul 50 ul C volume: ChargeSwitch Magnetic beads 30 ul 24 ul 18 ul 12 ul 6 ul D volume: ChargeSwitch Wash Buffer (W12) 1 mL 800 ul 600 ul 400 ul 200 ul ChargeSwitch Elution Buffer (E5) 50 ul 50 ul 50 ul 50 ul 50 ul
- # Before starting, mix Lysis buffer with proteinase K according to table above & scale up volume for multiple samples
- Preparing lysate
- - Add S volume of sample to 1.5mL tube
- - Add A volume + 30ul of Lysis mix (Lysis Buffer + proteinase K)
- - Add 5ul of RNase A
- - Pipette up & down 5x to mix
- - Incubate at RT for 20min to lyse the sample. Alternatively incubate on a rotating mixer
- Binding DNA
- - Vortex the tube containing ChargeSwitch Magnetic beads
- - Add B volume of ChargeSwitch Purification Buffer to the digested sample
- - Add 30 ul of ChargeSwitch Magnetic beads and pipette up & down 5x to mix (use p1000 pipette, avoid generating bubbles)
- - Incubate at RT for 2 min to allow DNA bind to magnetic beads
- - Place the sample tube in the MagnaRack for 3 min or until the bead forming a tight pellet. (I may use magnetic rack for 1.5mL tube available in the lab)
- - Carefully remove the spnt without disturbing the bead pellet
- Washing DNA
- - Remove sample tube from magnetic rack
- - Add D volume of ChargeSwitch Wash Buffer to the tube and pipette up & down 5x gently & avoid bubbles
- - Place the sample tube in the MagnaRack for 2 min or until the bead forming a tight pellet
- - Carefully remove the spnt without disturbing the bead pellet
- - Repeat washing step
- Eluting DNA
- - Remove sample tube from magnetic rack (there should be no spnt in the tube)
- - Add 50ul of ChargeSwitch Elution Buffer to the tube and pipette up & down gently 10x to resuspend magnetic beads
- # Do not use H2O for elution
- - Place the sample tube in the MagnaRack for 1 min or until the bead forming a tight pellet
- - Carefully transfer eluted DNA to 1.5mL tube
4. ZymoBead™ Genomic DNA kit[edit]
- -This protocol is for whole blood, serum and plasma samples
Procedures[edit]
- -Before starting, add beta-mercaptoethanol to Genomic Lysis Buffer to a final dilution of 0.5% (v/v). Vortex ZymoBead to resuspend
- Preparing lysate and binding DNA
- - Add 200ul of Genomic Lysis Buffer to 50ul samples in 1.5mL tube
- - Add 10ul ZymoBeads. Mix by inversion
- - Incubate at RT for 5 min
- - Centrifuge at 1,500xg for 1 min
- - Carefully remove the spnt without disturbing the bead pellet
- - Add 200ul of Genomic Lysis Buffer to the ZymoBeads
- Resuspend the beads by pipetting up & down
- Centrifuge at 1,500xg for 1 min
- - Carefully remove the spnt without disturbing the bead pellet
- Washing DNA
- - Add 200ul of DNA Pre-Wash Buffer to the ZymoBeads.
- - Resuspend the pellet, transfer to a new tube, centrifuge at 1,500xg for 1min
- - Carefully remove the spnt without disturbing the bead pellet
- Add 500ul of g-DNA Wash Buffer to the ZymoBeads.
- - Resuspend the pellet and centrifuge at 1,500xg for 1 min.
- - Carefully remove the spnt without disturbing the bead pellet
- Re-centrifuge briefly and remove the residual wash buffer
- Eluting DNA
- - Add >35ul of Elution Buffer or H2O and resuspend the pellet by pipetting up & down.
- - Centrifuge at 10,000xg for 1 min
- - Collect the spnt to the microcentrifuge tube.
Things to check[edit]
- - Beta-mercaptoethanol --> Yes
- - Magnetic rack for 1.5mL tube is working well? --> Yes
- - Need to use carriers?
- - ZymoBeads allows for 50ul samples. Do I need to do multiple tubes for total 200ul sample? Double check with Dr. Zhang the total volume required to test for each kit.
- - How to combine DNA purification with bisulfite conversion for bead-based methods.
- Recommended paper from Dr. Zhang: Bailey VJ et al, 2010
- - In this paper, the step of cell lysis and bisulfite conversion were performed in the same tube and used the same magnetic beads in both steps. The house made buffers of bisulfite conversion step were prepared to compatible with the magnetic beads used in this experiment.
- - BaileyVJ_etal2010_Supp_Methods
2014-03-04[edit]
- - Received 3 case samples from Sharmeela
Case/Accession # Project ID # Typr/Amount Daigonosis AA005 UCSD004.1 4-BC,P,S Eosinophilia AA007 UCSD004.2 4-BC,P,S Anemia AA008 UCSD004.3 4-BC,P, 3S Anemia
- Note: SampleUCSD004.3 had only 3 aliquots of serum
- - White caps : Buffy coat (BC)
- - Green caps: Plasma (P)
- - Blue caps : Serum (S)
2014-03-10[edit]
Discussion with Dr. Zhang
Next things to check[edit]
- - Waiting for Van (Dr. Sah's lab manager) for plate reader demonstration
- - Confirm with Dr. Zhang where can I use Bioanalyzer
- -> At Dr. Palsson's lab
- - Should I extract all samples (3 cases) or try out only one sample?
- -> Extract sample #1 & #2 and save #3
- - For sample #3, which has only 3 tube of serum, what are the three kits out of four I should use for DNA extraction?
- -> No need to extract sample #3 for now
- Continued on: [[1]]