Alice:Single/2015-4-30: Difference between revisions

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=SMARTer RNA seq protocol trial run #3=
=SMARTer RNA seq protocol trial run #3=
Last time I used Blue's old SMARTer kit to do a trial run, but the results weren't good.  
Last time I used Blue's old SMARTer kit to do a trial run, but the results weren't good.  
There are several possibilities where things might went wrong:  
There are several possibilities where things might went wrong:  
1) the primers of Blue's old kit were hydrolyzed/reagents weren't fresh;  
1) the primers of Blue's old kit were hydrolyzed/reagents weren't fresh;  
2) the amount was too little to be detected/seen on gel (didn't run QPCR);  
2) the amount was too little to be detected/seen on gel (didn't run QPCR);  
3) the technique wasn't good enough to get the experiments to work, more practice needed;
3) the technique wasn't good enough to get the experiments to work, more practice needed;
In order to make sure the reagents were of good quality, Song purchased a new SMARTer v3 kit to do a trial run [[ Link]]
In order to make sure the reagents were of good quality, Song purchased a new SMARTer v3 kit to do a trial run [[ Link]]


==Experimental Procedure==
==Experimental Procedure==
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#Thaw all the reagents needed for first-strand cDNA synthesis (except the enzyme) on ice. Gently vortex each reagent to mix and spin down briefly. Store on ice.
#Thaw all the reagents needed for first-strand cDNA synthesis (except the enzyme) on ice. Gently vortex each reagent to mix and spin down briefly. Store on ice.
#Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer - v3 with the RNase Inhibitor as indicated below (scale-up as needed):
#Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer - v3 with the RNase Inhibitor as indicated below (scale-up as needed):
19 μl     10X Lysis Buffer - v3 RNase
{| {{table}}
  1 μl     Inhibitor  
| align="center" style="background:#f0f0f0;"|'''19 μl'''
20 μl     Total Volume
| align="center" style="background:#f0f0f0;"|'''10X Lysis Buffer v3'''
|-
| 1 μl||Rnase Inhibitor
|-
| 20 μl||Total Volume
|}
  Vortex briefly to mix, then spin down
  Vortex briefly to mix, then spin down
#Prepare each sample (10 μl total volume) in individual 0.2 ml RNase-free PCR tubes or in an 8-well strip.
#Prepare each sample (10 μl total volume) in individual 0.2 ml RNase-free PCR tubes or in an 8-well strip.
Line 26: Line 31:
| align="center" style="width:80px;background:#f0f0f0;"|'''test sample'''
| align="center" style="width:80px;background:#f0f0f0;"|'''test sample'''
|-
|-
| 10X Reaction Buffer||1 ul ||1 ul ||1 ul ||
| 10X Reaction Buffer||1 ul ||1 ul ||1 ul
|-
|-
| Nuclease-free water||9 ul ||0-8 ul ||0-8 ul ||
| Nuclease-free water||9 ul ||0-8 ul ||0-8 ul
|-
|-
| Diluted Control RNA||-||1-9 ul || -||
| Diluted Control RNA||-||1-9 ul || -
|-
|-
| Sample||-||-||1-9 ul ||
| Sample||-||-||1-9 ul
|-
|-
| Total colvme||10 ul || 10 ul || 10 ul ||
| Total colvme||10 ul || 10 ul || 10 ul
|}
|}
#Place the samples on ice, and add 1 μl of 3’ SMART CDS Primer II A (12 μM). Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube:
#Place the samples on ice, and add 1 μl of 3’ SMART CDS Primer II A (12 μM). Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube:
10 μl   Cell/Total RNA in Reaction Buffer (from Table above)
{| {{table}}
  1 μl   3’ SMART CDS Primer II A (12 μM)
| align="center" style="background:#f0f0f0;"|'''10 μl'''
11 μl   Total Volume
| align="center" style="background:#f0f0f0;"|'''Cell/Total RNA in Reaction Buffer'''
 
|-
| 1 μl||3’ SMART CDS Primer II A (12 μM)
|-
| 11 μl||Total Volume
|}
#Place the tubes into a preheated thermal cycler and run 72°C 3 min  then 4°C forever
#Place the tubes into a preheated thermal cycler and run 72°C 3 min  then 4°C forever
#When the thermal cycler reaches 4°C, remove the tubes from the thermal cycler and put them on ice.
#When the thermal cycler reaches 4°C, remove the tubes from the thermal cycler and put them on ice.
#Preheat the thermal cycler to 42°C.
#Preheat the thermal cycler to 42°C.
#Meanwhile, prepare enough Master Mix for all the reactions, plus 10% of the total reaction mix volume, by combining the following reagents in the order shown at room temperature:
#Meanwhile, prepare enough Master Mix for all the reactions, plus 10% of the total reaction mix volume, by combining the following reagents in the order shown at room temperature:
  4    μl         5X First-Strand Buffer
{| {{table}}
  0.5 μl         DTT (100 mM)
| align="center" style="background:#f0f0f0;"|'''4    μl'''
  1    μl       dNTP Mix (20 mM)
| align="center" style="background:#f0f0f0;"|'''5X First-Strand Buffer'''
  1    μl       SMARTer IIA Oligonucleotide (12 μM)
|-
  0.5 μl       RNase Inhibitor (40 U/μl)
| 0.5 μl||DTT (100 mM)
  2    μl       SMARTScribe Reverse Transcriptase (100 U/μl)*  
|-
  9    μl       Total Volume added per reaction
| 1    μl||dNTP Mix (20 mM)
|-
| 1    μl||SMARTer IIA Oligonucleotide (12 μM)
|-
| 0.5 μl||RNase Inhibitor (40 U/μl)
|-
| 2    μl||SMARTScribe Reverse Transcriptase (100 U/μl)*
|-
| 9    μl||Total Volume added per reaction
|}
  Add the reverse transcriptase to the Master Mix just prior to use, making sure to gently mix the reverse transcriptase tube without vortexing before adding it.  
  Add the reverse transcriptase to the Master Mix just prior to use, making sure to gently mix the reverse transcriptase tube without vortexing before adding it.  
  Mix the Master Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube.
  Mix the Master Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube.
Line 57: Line 75:
#Add 9 μl of the Master Mix to each reaction tube. Mix the contents of the tubes by gently pipetting, and spin them briefly to collect the contents at the bottom.
#Add 9 μl of the Master Mix to each reaction tube. Mix the contents of the tubes by gently pipetting, and spin them briefly to collect the contents at the bottom.
#Place the tubes in a thermal cycler with a heated lid, preheated to 42°C. Run the following program:
#Place the tubes in a thermal cycler with a heated lid, preheated to 42°C. Run the following program:
42°C 90 min  
42°C 90 min  -> 70°C 10 min -> 15°C forever
  70°C 10 min  
STOPPING POINT: The tubes can be stored at 4°C overnight.
15°C forever
STOPPING POINT: The tubes can be stored at 4°C overnight.


===cDNA amplification by LD PCR===
===cDNA amplification by LD PCR===
Line 66: Line 82:
#Prepare enough PCR Master Mix for all the reactions, plus 10% of the total reaction mix volume. Combine the following reagents in the order shown.
#Prepare enough PCR Master Mix for all the reactions, plus 10% of the total reaction mix volume. Combine the following reagents in the order shown.
NOTE: Remove the DNA polymerase from the freezer, gently mix the tube without vortexing, and add to the Master Mix just before use. Mix the Master Mix well by vortexing gently and spin the tube briefly in a microcentrifuge to collect the contents at the bottom of the tube:
NOTE: Remove the DNA polymerase from the freezer, gently mix the tube without vortexing, and add to the Master Mix just before use. Mix the Master Mix well by vortexing gently and spin the tube briefly in a microcentrifuge to collect the contents at the bottom of the tube:
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''25 ul'''
| align="center" style="background:#f0f0f0;"|'''2X SeqAmp PCR Buffer'''
|-
| 1 ul||PCR Primer II A - v3 (12 μM)
|-
| 1 ul|| Nuclease-free water
|-
| 3 ul|| SeqAmp DNA Polymerase
|-
| 30 ul||Total Volume added per reaction
|}
#Add 30 μl of PCR Master Mix to each tube containing 20 μl first-strand cDNA product from Section IV.E. Mix well and briefly spin in a minicentrifuge to collect the contents at the bottom of the tube(s).
#Add 30 μl of PCR Master Mix to each tube containing 20 μl first-strand cDNA product from Section IV.E. Mix well and briefly spin in a minicentrifuge to collect the contents at the bottom of the tube(s).
#Place the tube(s) in a preheated thermal cycler with a heated lid and run the following program:
#Place the tube(s) in a preheated thermal cycler with a heated lid and run the following program:
95°C 1 min -> ( 98°C for 10 sec -> 65°C for 30 sec -> 68°C for 3 min ) -> 72°C 10 min
95°C 1 min -> ( 98°C for 10 sec -> 65°C for 30 sec -> 68°C for 3 min ) -> 72°C 10 min



Revision as of 18:32, 30 April 2015

SMARTer RNA seq protocol trial run #3

Last time I used Blue's old SMARTer kit to do a trial run, but the results weren't good. There are several possibilities where things might went wrong: 1) the primers of Blue's old kit were hydrolyzed/reagents weren't fresh; 2) the amount was too little to be detected/seen on gel (didn't run QPCR); 3) the technique wasn't good enough to get the experiments to work, more practice needed; In order to make sure the reagents were of good quality, Song purchased a new SMARTer v3 kit to do a trial run Link

Experimental Procedure

  • The Control RNA is supplied at a concentration of 1 μg/μl. It should be diluted in nuclease-free water to match the concentration of test sample.
  • Perform serial dilutions on the Control RNA to appropriate concentration.

First strand cDNA Synthesis

  1. Thaw all the reagents needed for first-strand cDNA synthesis (except the enzyme) on ice. Gently vortex each reagent to mix and spin down briefly. Store on ice.
  2. Prepare a stock solution of 10X Reaction Buffer by mixing the 10X Lysis Buffer - v3 with the RNase Inhibitor as indicated below (scale-up as needed):
19 μl 10X Lysis Buffer v3
1 μl Rnase Inhibitor
20 μl Total Volume
Vortex briefly to mix, then spin down
  1. Prepare each sample (10 μl total volume) in individual 0.2 ml RNase-free PCR tubes or in an 8-well strip.
    1. If working with purified total RNA, transfer 1–9 μl to a 0.2 ml RNase-free PCR tube. Bring the volume to 9 μl with nuclease-free water. Add 1 μl of 10X Reaction Buffer.
    2. If working with cells, isolate cells in validated media and transfer to a 0.2 ml RNase-free PCR tube. Bring the volume to 9 μl with nuclease-free water. Add 1 μl 10X Reaction Buffer. Gently vortex or pipette to mix the sample. Incubate at room temperature for 5 minutes.
Components negative control positive control test sample
10X Reaction Buffer 1 ul 1 ul 1 ul
Nuclease-free water 9 ul 0-8 ul 0-8 ul
Diluted Control RNA - 1-9 ul -
Sample - - 1-9 ul
Total colvme 10 ul 10 ul 10 ul
  1. Place the samples on ice, and add 1 μl of 3’ SMART CDS Primer II A (12 μM). Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube:
10 μl Cell/Total RNA in Reaction Buffer
1 μl 3’ SMART CDS Primer II A (12 μM)
11 μl Total Volume
  1. Place the tubes into a preheated thermal cycler and run 72°C 3 min then 4°C forever
  2. When the thermal cycler reaches 4°C, remove the tubes from the thermal cycler and put them on ice.
  3. Preheat the thermal cycler to 42°C.
  4. Meanwhile, prepare enough Master Mix for all the reactions, plus 10% of the total reaction mix volume, by combining the following reagents in the order shown at room temperature:
4 μl 5X First-Strand Buffer
0.5 μl DTT (100 mM)
1 μl dNTP Mix (20 mM)
1 μl SMARTer IIA Oligonucleotide (12 μM)
0.5 μl RNase Inhibitor (40 U/μl)
2 μl SMARTScribe Reverse Transcriptase (100 U/μl)*
9 μl Total Volume added per reaction
Add the reverse transcriptase to the Master Mix just prior to use, making sure to gently mix the reverse transcriptase tube without vortexing before adding it. 
Mix the Master Mix well by gently vortexing and then spin the tube(s) briefly in a minicentrifuge to collect the contents at the bottom of the tube.
  1. Add 9 μl of the Master Mix to each reaction tube. Mix the contents of the tubes by gently pipetting, and spin them briefly to collect the contents at the bottom.
  2. Place the tubes in a thermal cycler with a heated lid, preheated to 42°C. Run the following program:

42°C 90 min -> 70°C 10 min -> 15°C forever STOPPING POINT: The tubes can be stored at 4°C overnight.

cDNA amplification by LD PCR

  1. Thaw all the reagents needed for PCR (except the enzyme) on ice. Gently vortex each reagent tube to mix and spin down briefly. Store on ice.
  2. Prepare enough PCR Master Mix for all the reactions, plus 10% of the total reaction mix volume. Combine the following reagents in the order shown.

NOTE: Remove the DNA polymerase from the freezer, gently mix the tube without vortexing, and add to the Master Mix just before use. Mix the Master Mix well by vortexing gently and spin the tube briefly in a microcentrifuge to collect the contents at the bottom of the tube:

25 ul 2X SeqAmp PCR Buffer
1 ul PCR Primer II A - v3 (12 μM)
1 ul Nuclease-free water
3 ul SeqAmp DNA Polymerase
30 ul Total Volume added per reaction
  1. Add 30 μl of PCR Master Mix to each tube containing 20 μl first-strand cDNA product from Section IV.E. Mix well and briefly spin in a minicentrifuge to collect the contents at the bottom of the tube(s).
  2. Place the tube(s) in a preheated thermal cycler with a heated lid and run the following program:

95°C 1 min -> ( 98°C for 10 sec -> 65°C for 30 sec -> 68°C for 3 min ) -> 72°C 10 min