mRNA Sample preparation Track Form
( Homebrew with NEB Kit Sample Preparation)
Sample QC:
Date: _____________________
1, The concentration of RNA
Sample ID
Bioanalyzer Result
Con.(ng/ul)
2, The quality of RNA
Page 1 of 4
HomeBrew GA Sample Preparation Date:__________ SampleID:________________
1 mRNA purification and Fragmentation:
Date :_____________________
Following the Illumina mRNA prepping protocol to purify the mRNA and elute the mRNA
in 130 ul DEPC water and then go to shear with Covarius.
___300_bp shearing (Covaris)
Duty Cycle:
10%
Intensity
5
cycles Per Burst
200
Time
35
__2__Cycles
After shearing, clean up the sample with Ethanol. Mix the following components in a
sterile 1.5ml microcentrifuge tube:
Reagent
Volume (ul)
130
13
3
390
536
Fragmented RNA from shearing
3M Sodium Acetate pH5.20
Linear Acrylamide, 10mg/ml
100% Ethanol
Total Volume
Incubate at -80°C or in a dry ice/methanol bath for at least 2 hour
1, Centrifuge at 14,000 rpm for 25 minutes at 4°C in a microcentrifuge.
2, Carefully remove ethanol.
3. Wash pellet with 300 μl of freshly prepared 70% ethanol. Carefully pipette up and
down the pellet. Make sure the pellet does not get stuck in the tip.
4, Centrifuge at 14,000 rpm for 5 minutes at 4°C in a microcentrifuge. Carefully remove
all 70% ethanol.
5, Repeat steps 5 and 6 twice.
6, Air dry pellet for up to 10 minutes at room temperature (or longer if necessary) to
remove residual ethanol.
7, Resuspend in 14.5 μl Nuclease-free Water.
Page 2 of 4
HomeBrew GA Sample Preparation Date:__________ SampleID:________________
2, cDNA Synthesis
First Strand cDNA Synthesis
1. Mix the following components in a sterile PCR tube:
Reagent
Volume (ul)
Fragmented mRNA
Random Primers
Total Volume
13.5
1
14.5
2. Incubate in a preheated thermal cycler for 5 minutes at 65C.
3. Spin tube briefly and place on ice.
4. To the fragmented mRNA and Random Primers add:
Reagent
Volume (ul)
4
0.5
19
5X First Strand Synthesis Reaction Buffer
Murine RNase Inhibitor
Total Volume
5. Incubate in a preheated thermal cycler for 2 minutes at 25°C.
6. Add 1 μl ProtoScript II Reverse Transcriptase to the reaction.
7. Incubate sample as follows:
10 minutes at 25°C
50 minutes at 42°C
15 minutes at 70°C
Hold at 4°C
8. Place the tube on ice.
Second Strand cDNA Synthesis
1. Add the following reagents to the First Strand Synthesis reaction:
Reagent
Volume (ul)
Nuclease-free Water
10X Second Strand Synthesis Reaction Buffer
Second Strand Synthesis Enzyme Mix
Total Volume
48
8
4
60
2. Mix thoroughly by gentle pipetting.
3. Incubate in a thermal cycler for 2.5 hours at 16°C.
Page 3 of 4
HomeBrew GA Sample Preparation Date:__________ SampleID:________________
3 Final Library Prep.
1. End Repair:
Date: _______________
Date:__________
Reagents
DNA sample
10X T4 ligase buffer with 10mM ATP
10mM dNTPmix
T4 DNA Polymerase
DNA Polymerase I Klenow LG Fragment
T4 PNK
Ultra Pure Water
Vol. per sample (μl)
50
10
4
2
1
1
32
Incubate at 20ºC for 30 minutes. QIAquick PCR purify, elute in 34ul.
2. Add ‘A’ Bases to 3’ End:
Reagents
DNA sample
10X NEBuffer 2
1mM dATP
Klenow Fragment ( exo-)
Total Volume
Date: _______________
Vol. per sample (μl)
34
5
10
1
50
Incubate at 37ºC for 30 minutes. QIAquick MinElute, elute in 10ul.
3. Ligate Adapters to DNA Fragments:
Reagents
Date: __________
Vol. per sample (ul)
DNA sample
T4 DNA ligase buffer
Water
Adapter Oligo Mix *
T4 DNA ligase
10
5
20
10
5
*Note: Adaptor: (PE Adapter Oligo Mix or Genomic adapter (SR) ) (Lot:__________)
Incubate at room temperature for 30 minutes.
QIAquick MinElute, elute in 10ul.
4. Size-Selection:
Date: ____________
Using Ampure Beads to do the size-selection: follow the 400bp size selection protocol.
Page 4 of 4
HomeBrew GA Sample Preparation Date:__________ SampleID:________________
5. PCR Amplification:
Date: ____________
Reagents
Water
Library DNA Template
Library Amplication Master Mix
Primer 1
Primer 2
Total
Vol. per sample (μl)
10.5
1
12.5
0.5
0.5
25
PCR condition:
98ºC for 30 seconds
98ºC for 10 seconds
65ºC for 30 seconds
72ºC for 30 seconds
72ºC for 5 minutes
Gel Qiaquick purify, elute in 30ul.
6.Validate the Library:
Date: ____________
Quantify the library using Invitrogen’s Qubit Fluorometer and the associated Quant-iTTM
dsDNA BR assay kit according to manufacturer’s instructions.
Sample ID
Qubit assay (μg/ml)
Page 5 of 4
HomeBrew GA Sample Preparation Date:__________ SampleID:________________
10X