Noi/NOTES/2014-3-11: Difference between revisions
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: # 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 '''200ul''' of '''plasma/serum''' to 1.5mL tube. | :: - Add '''200ul''' of '''plasma/serum/buffy coat''' to 1.5mL tube. | ||
:::{| {{table}} class = wikitable | :::{| {{table}} class = wikitable | ||
| align="center" style="background:#f0f0f0;"|'''Samples''' | | align="center" style="background:#f0f0f0;"|'''Samples''' | ||
Line 138: | Line 138: | ||
::: - Eluted DNA from buffy coat samples was red because erythrocytes were carried over with buffy coat layer either in sample #1 or #2. | ::: - Eluted DNA from buffy coat samples was red because erythrocytes were carried over with buffy coat layer either in sample #1 or #2. | ||
:: - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol --> ''I skipped this step by drying the column for 3 min before eluting DNA.'' | :: - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol --> ''I skipped this step by drying the column for 3 min before eluting DNA.'' | ||
---- | |||
: - For bead-based method, I will split samples for two processes. One for regular DNA extraction and one will go to bisulfite treatment after DNA binding to beads without washing and elution. | |||
: - The volume of sample was not exactly 200ul (plasma and buffy coat ~100ul for each, serum ~200ul), so I used different volume for DNA extraction (50, 100 or 200ul) | |||
=== 3. ChargeSwitch® gDNA 1 ml Serum kit === | |||
==== Procedures ==== | |||
:: - Add plasma and sample to 1.5mL tube (adjust to total 200ul). | |||
:: - Note: the volume of sample could be up to 1000ul | |||
::{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''1X PBS (ul)''' | |||
|- | |||
| #1-P-CS||50.00||150.00 | |||
|- | |||
| #1-S-CS||100.00||100.00 | |||
|- | |||
| #1-BC-CS||50.00||150.00 | |||
|- | |||
| #2-P-CS||50.00||150.00 | |||
|- | |||
| #2-S-CS||100.00||100.00 | |||
|- | |||
| #2-BC-CS||50.00||150.00 | |||
|} | |||
: - Mix 910ul of Lysis Buffer + 195 Proteinase K (140:30, 2 tubes) | |||
:: - Add '''170ul of Lysis Buffer and Proteinase K mix soulution (140ul of Lysis Buffer and 30ul of Proteinase K for 200ul sample) | |||
:: - Pipette up & down 5x to mix | |||
:: - Incubate at RT for 20min to lyse the sample. | |||
: <u>Binding DNA</u> | |||
: - Vortex the tube containing ChargeSwitch Magnetic beads | |||
: - Mix 50ul of ChargeSwitch Purification Buffer + 6ul of ChargeSwitch Magnetic beads for 13 rxn (650ul buffer + 78ul beads) | |||
:: - Add 56ul of ChargeSwitch Purification Buffer and ChargeSwitch Magnetic beads mix to the digested sample | |||
:: - 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 | |||
: <u>Washing DNA</u> | |||
:: - Remove sample tube from magnetic rack | |||
:: - Add 200ul 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 | |||
: <u>Eluting DNA</u> | |||
:: - 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 | |||
---- | |||
'''<u>For bisulfite treatment</u>''' | |||
:: - After DNA binding step prior to washing step, I resuspend the beads with 20ul of ChargeSwitch Elution Buffer | |||
:: - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X | |||
:: - Transfer solution to 200ul tube | |||
:: - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit | |||
:: - Elute with 17ul of Elution buffer --> volume after spin is ~16ul | |||
:: - Used 3ul for Qubit ssDNA assay | |||
:: - Note: This kit was performed after Zymobeads. I saw that 2ul for assay was not enough to be detected for some samples, so I increase the sample volume from 2ul to 3ul. | |||
:: - I will calculate total amount in 16ul --> normalize yield to 100ul input sample | |||
<u>Quant-iT ssDNA</u> | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''uL used''' | |||
| align="center" style="background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample Conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Yield in 16ul (ng)''' | |||
|- | |||
| #1-P-CS||<1ng/mL||||||||||||NA | |||
|- | |||
| #1-S-CS||<1ng/mL||||||||||||NA | |||
|- | |||
| #1-BC-CS||1.2||ng/mL||3||66.7||79.00||pg/ul||1.26 | |||
|- | |||
| #2-P-CS||<1ng/mL||||||||||||NA | |||
|- | |||
| #2-S-CS||3.1||ng/mL||3||66.7||210.00||pg/ul||3.36 | |||
|- | |||
| #2-BC-CS||64.6||ng/mL||3||66.7||4.31||ng/ul||68.96 | |||
|} | |||
<u>Normalized to 100ul sample input</u> | |||
:{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume used (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''yields in 100ul (ng)''' | |||
|- | |||
| #1-P-CS||50||||NA | |||
|- | |||
| #1-S-CS||100||||NA | |||
|- | |||
| #1-BC-CS||50||1.26||2.53 | |||
|- | |||
| #2-P-CS||50||||NA | |||
|- | |||
| #2-S-CS||100||3.36||3.36 | |||
|- | |||
| #2-BC-CS||50||68.96||137.92 | |||
|} | |||
<br> | |||
==== 4. ZymoBead™ Genomic DNA kit ==== | |||
==== Procedures ==== | |||
: -Before starting, add 250ul of beta-mercaptoethanol to 50mL of Genomic Lysis Buffer to a final dilution of 0.5% (v/v). Vortex ZymoBead to resuspend | |||
: <u>Preparing lysate and binding DNA</u> | |||
:: - 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 | |||
: <u>Washing DNA</u> | |||
:: - 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 | |||
: <u>Eluting DNA</u> | |||
:: - Add '''>35ul''' of '''Elution Buffer''' and resuspend the pellet by pipetting up & down. | |||
:: - Centrifuge at 10,000xg for 1 min | |||
:: - Collect the spnt to the 1.5mL tube tube. | |||
---- | |||
'''<u>For bisulfite treatment</u>''' | |||
:: - After DNA binding step prior to pre-washing step, I resuspend the beads with 20ul of Elution Buffer | |||
:: - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X | |||
:: - Transfer solution to 200ul tube | |||
:: - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit | |||
:: - Elute with 17ul of Elution buffer --> volume after spin is ~16ul | |||
:: - Used 2ul for Qubit ssDNA assay | |||
:: - I will calculate total amount in 16ul --> normalize yield to 100ul input sample | |||
<u>Quant-iT ssDNA</u> | |||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''uL used''' | |||
| align="center" style="background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample Conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Yield in 16ul (ng)''' | |||
|- | |||
| #1-P-Z||<1ng/mL|||||||||||| | |||
|- | |||
| #1-S-Z||1.3||ng/mL||2||100||130.00||pg/ul||1.82 | |||
|- | |||
| #1-BC-Z||12.6||ng/mL||2||100||1.26||ng/ul||17.64 | |||
|- | |||
| #2-P-Z||1.5||ng/mL||2||100||150.00||pg/ul||2.10 | |||
|- | |||
| #2-S-Z||3.1||ng/mL||2||100||310.00||pg/ul||4.34 | |||
|- | |||
| #2-BC-Z||38||ng/mL||2||100||3.80||ng/ul||53.20 | |||
|} | |||
<u>Normalized to 100ul sample input</u> | |||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume used (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''yields in 100ul (ng)''' | |||
|- | |||
| #1-P-Z||50||NA||NA | |||
|- | |||
| #1-S-Z||50||1.82||3.64 | |||
|- | |||
| #1-BC-Z||50||17.64||35.28 | |||
|- | |||
| #2-P-Z||50||2.10||4.2 | |||
|- | |||
| #2-S-Z||50||4.34||8.68 | |||
|- | |||
| #2-BC-Z||50||53.20||106.4 | |||
|} |
Revision as of 15:55, 13 March 2014
MONOD test on human plasma/serum
- Continued from: [[1]]
Sample labeling
' | Labeling on original tubes | Labeling on working tubes | ||||
Sample ID | Plasma (P) | Serum (S) | Buffy coat (BC) | Plasma (P) | Serum (S) | Buffy coat (BC) |
UCSD004.1 | UCSD004#1-P-Q | UCSD004#1-S-Q | UCSD004#1-P-Q | #1-P-Q | #1-S-Q | #1-P-Q |
UCSD004#1-P-Nu | UCSD004#1-S-Nu | UCSD004#1-P-Nu | #1-P-Nu | #1-S-Nu | #1-P-Nu | |
UCSD004#1-P-CS | UCSD004#1-S-CS | UCSD004#1-P-CS | #1-P-CS | #1-S-CS | #1-P-CS | |
UCSD004#1-P-Z | UCSD004#1-S-Z | UCSD004#1-P-Z | #1-P-Z | #1-S-Z | #1-P-Z | |
UCSD004.2 | UCSD004#2-P-Q | UCSD004#2-S-Q | UCSD004#2-P-Q | #2-P-Q | #2-S-Q | #2-P-Q |
UCSD004#2-P-Nu | UCSD004#2-S-Nu | UCSD004#2-P-Nu | #2-P-Nu | #2-S-Nu | #2-P-Nu | |
UCSD004#2-P-CS | UCSD004#2-S-CS | UCSD004#2-P-CS | #2-P-CS | #2-S-CS | #2-P-CS | |
UCSD004#2-P-Z | UCSD004#2-S-Z | UCSD004#2-P-Z | #2-P-Z | #2-S-Z | #2-P-Z |
Order in sample IDs/codes | Description |
1 | Project ID |
2 | # of sample (1,2,3,…,N) |
3 | Type of blood fraction |
4 | Extracted by kit |
P | Plasma |
S | Serum |
BC | Buffy coat |
Q | Extracted by QIAGEN kit |
Nu | Extract by nucleospin column |
CS | Extracted by ChargeSwitch kit |
Z | Extracted by Zymo kit |
Serum/Plasma DNA extraction
- - For column-based method, I will use 200ul of plasma/serum/buffy coat
1. QIAamp DNA Blood Mini Kit
- - Follow protocol described in [[2]]
- - Resuspend lyophilized QIAGEN Protease with 1.2 ml protease solvent.
Procedures
- 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
- - Note: All samples were provided at different volumes, I used different volumes for DNA extraction. At the end, the yields by each method will be compared with normalized sample volume.
Samples Volume (ul) 1X PBS (ul) #1-P-Q 100.00 100.00 #1-S-Q 200.00 0.00 #1-BC-Q 100.00 100.00 #2-P-Q 150.00 50.00 #2-S-Q 200.00 0.00 #2-BC-Q 100.00 100.00
- - 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). --> I eluted with 26ul Buffer AE
- - Incubate at RT for 5 min & centrifuge at 6,000xg for 1 min
2. NucleoSpin® Plasma XS kit
- - Follow protocol described in [[3]]
- - Add 1.35mL of Proteinase Buffer (PB) to 30mg of lyophilized Proteinase K for 50 prep kit.
Procedures
- # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min
- Preparing lysate
- - Add 200ul of plasma/serum/buffy coat to 1.5mL tube.
Samples Volume (ul) 1X PBS (ul) #1-P-Nu 100.00 100.00 #1-S-Nu 200.00 0.00 #1-BC-Nu 100.00 100.00 #2-P-Nu 200.00 0.00 #2-S-Nu 150.00 50.00 #2-BC-Nu 100.00 100.00
- - Add 20ul of proteinase K to plasma/serum sample
- - Mix and incubate at 37C for 10min
- Binding DNA
- - Add 1.5 volume (300ul) of Buffer BB (keep sample:binding buffer ratio 1:1.5 v/v)
- - 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 26ul of Elution buffer to the column (range 5-30ul)
- - Centrifuge at 11,000xg for 30s
- - Eluted DNA from buffy coat samples was red because erythrocytes were carried over with buffy coat layer either in sample #1 or #2.
- - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol --> I skipped this step by drying the column for 3 min before eluting DNA.
- - For bead-based method, I will split samples for two processes. One for regular DNA extraction and one will go to bisulfite treatment after DNA binding to beads without washing and elution.
- - The volume of sample was not exactly 200ul (plasma and buffy coat ~100ul for each, serum ~200ul), so I used different volume for DNA extraction (50, 100 or 200ul)
3. ChargeSwitch® gDNA 1 ml Serum kit
Procedures
- - Add plasma and sample to 1.5mL tube (adjust to total 200ul).
- - Note: the volume of sample could be up to 1000ul
Samples Volume (ul) 1X PBS (ul) #1-P-CS 50.00 150.00 #1-S-CS 100.00 100.00 #1-BC-CS 50.00 150.00 #2-P-CS 50.00 150.00 #2-S-CS 100.00 100.00 #2-BC-CS 50.00 150.00
- - Mix 910ul of Lysis Buffer + 195 Proteinase K (140:30, 2 tubes)
- - Add 170ul of Lysis Buffer and Proteinase K mix soulution (140ul of Lysis Buffer and 30ul of Proteinase K for 200ul sample)
- - Pipette up & down 5x to mix
- - Incubate at RT for 20min to lyse the sample.
- Binding DNA
- - Vortex the tube containing ChargeSwitch Magnetic beads
- - Mix 50ul of ChargeSwitch Purification Buffer + 6ul of ChargeSwitch Magnetic beads for 13 rxn (650ul buffer + 78ul beads)
- - Add 56ul of ChargeSwitch Purification Buffer and ChargeSwitch Magnetic beads mix to the digested sample
- - 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 200ul 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
For bisulfite treatment
- - After DNA binding step prior to washing step, I resuspend the beads with 20ul of ChargeSwitch Elution Buffer
- - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X
- - Transfer solution to 200ul tube
- - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit
- - Elute with 17ul of Elution buffer --> volume after spin is ~16ul
- - Used 3ul for Qubit ssDNA assay
- - Note: This kit was performed after Zymobeads. I saw that 2ul for assay was not enough to be detected for some samples, so I increase the sample volume from 2ul to 3ul.
- - I will calculate total amount in 16ul --> normalize yield to 100ul input sample
Quant-iT ssDNA
Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. Unit Yield in 16ul (ng) #1-P-CS <1ng/mL NA #1-S-CS <1ng/mL NA #1-BC-CS 1.2 ng/mL 3 66.7 79.00 pg/ul 1.26 #2-P-CS <1ng/mL NA #2-S-CS 3.1 ng/mL 3 66.7 210.00 pg/ul 3.36 #2-BC-CS 64.6 ng/mL 3 66.7 4.31 ng/ul 68.96
Normalized to 100ul sample input
Sample Volume used (ul) Yields (ng) yields in 100ul (ng) #1-P-CS 50 NA #1-S-CS 100 NA #1-BC-CS 50 1.26 2.53 #2-P-CS 50 NA #2-S-CS 100 3.36 3.36 #2-BC-CS 50 68.96 137.92
4. ZymoBead™ Genomic DNA kit
Procedures
- -Before starting, add 250ul of beta-mercaptoethanol to 50mL of 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 and resuspend the pellet by pipetting up & down.
- - Centrifuge at 10,000xg for 1 min
- - Collect the spnt to the 1.5mL tube tube.
For bisulfite treatment
- - After DNA binding step prior to pre-washing step, I resuspend the beads with 20ul of Elution Buffer
- - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X
- - Transfer solution to 200ul tube
- - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit
- - Elute with 17ul of Elution buffer --> volume after spin is ~16ul
- - Used 2ul for Qubit ssDNA assay
- - I will calculate total amount in 16ul --> normalize yield to 100ul input sample
Quant-iT ssDNA
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Sample Conc. | Unit | Yield in 16ul (ng) |
#1-P-Z | <1ng/mL | ||||||
#1-S-Z | 1.3 | ng/mL | 2 | 100 | 130.00 | pg/ul | 1.82 |
#1-BC-Z | 12.6 | ng/mL | 2 | 100 | 1.26 | ng/ul | 17.64 |
#2-P-Z | 1.5 | ng/mL | 2 | 100 | 150.00 | pg/ul | 2.10 |
#2-S-Z | 3.1 | ng/mL | 2 | 100 | 310.00 | pg/ul | 4.34 |
#2-BC-Z | 38 | ng/mL | 2 | 100 | 3.80 | ng/ul | 53.20 |
Normalized to 100ul sample input
Sample | Volume used (ul) | Yields (ng) | yields in 100ul (ng) |
#1-P-Z | 50 | NA | NA |
#1-S-Z | 50 | 1.82 | 3.64 |
#1-BC-Z | 50 | 17.64 | 35.28 |
#2-P-Z | 50 | 2.10 | 4.2 |
#2-S-Z | 50 | 4.34 | 8.68 |
#2-BC-Z | 50 | 53.20 | 106.4 |