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Setting up the real-time RT reaction
This spreadsheet is designed to make running PCRs easier. Below is a list of the basic supplies you need to run
your PCRs, along with current list prices. On the following pages, enter your text into the gray boxes, and the
spreadsheet will calculate the items you need to add for you. If you have questions, email me at
gray@eohsi.rutgers.edu.
Save this file as a template in excel on your computer.
Enter your values on the next four pages into the gray boxes to make the master mixes.
Promega
2800 Woods Hollow Road
Madison WI 53711
Requirements: UNITED STATES
Tel: 1-608-274-4330
Fax: 1-608-277-2516
CAT# Name Amount # Cost Total Toll free Tel: 1-800-356
Promega Toll free Fax: 1-800-356
N2615 Rnasin 10000U 2 254 508 E-mail Address: custserv@promega
E-mail Address: techserv@promega
Web Address: www.promega.com
Applied Biosystems
5ml
4309155 SYBR Green master mix 20 349 6980 Applied Biosystems
Division Headquarters
850 Lincoln Centre Drive
Foster City
Invitrogen CA 94404
18080-085 Superscript III RT 4x10000 4 720 2880 U.S.A.
18427-013 Nucleotide mix 2 307 614 Phone: 1 650-638-5800, 1 800
48190-011 Random primers 103 Fax: 1 650-638-5884
E-mail: Please see the Contact Us
VWR
Versagene RNA prep kit
VGR-O250C 2 810 1620
invitrogen VWR
1600 Faraday Avenue 1310 Goshen Parkway
PO Box 6482 West Chester, PA 19380
Carlsbad, California 92008 Orders: 1-800-932-5000
Phone: (760) 603-7200 Web Orders: www.vwr.com
FAX: (760) 602-6500 Phone: (610) 431-1700
Fax: (610)431-9174
Random hexamers: I make these myself, by ordering them from IDT. Here's how:
Order a larger size quantity of hexamers. They are cheap, since they are only 6 nucleotides long.
Order each of these: NNNNNA, NNNNNC, NNNNNG, and NNNNNT. (they won't let your order NNNNNN)!
Dilute each to 2 ug/uL.
Mix together equal concentrations of all four.
Now you have 500 ng/uL random hexamers!
Dual labeled probes/TaqMan-style PCRs
If you find published primers in a paper for TaqMan style PCR, you can use a
dual-labeled probe from IDT to match the TaqMan PCR, without having to buy
the TaqMan kit. Order the two outside primers like normal. For the inside
probe, order a dual-labeled probe, such as 5'FAM and 3'BHQ. These are
approximately $150 from IDT (www.idtdna.com). Use the TaqMan PCR
reaction recipe as listed below.
CAT# Name Amount # Cost Total
Qiagen
205213 Sensiscript 200 kit 1 722
205211 Sensiscript 50 kit 1 205
you need to run
oxes, and the
oods Hollow Road
n WI 53711
4330
2516
800-356-9526
800-356-1970
Address: custserv@promega.com
Address: techserv@promega.com
ddress: www.promega.com
Biosystems
n Headquarters
incoln Centre Drive
638-5800, 1 800-327-3002
5884
l: Please see the Contact Us Section
11/26/2012 C:\Docstoc\Working\pdf\bff6c6be-9db3-4b89-9254-5690ec7fc8a4.xlsx
Project: insert project name/lab book # here
Joshua Gray's Applied Biosystems Calculator Instructions:
Add your value
in the yellow bo
Enter your Numbers Here: mixes: 1 & 2.
the sheet.
Number samples 54
RT Reaction Volume 30 (usually 20, enough for 20 PCRs) FAQ
RNA volume 15 (up to 10 in a 20uL reaction) How much RN
10 ug. Howeve
superscript. Fo
recommend Se
Master mix Stock
Per reaction I
Per Reaction
10X RT buffer 3 178.2
25X dNTP mix (100 mM) 1.2 71.28
10X RT random primers 3 178.2
Rnasin 1.5 89.1
Multiscribe RT 1.5 89.1
nuclease free water 4.8 285.12
Total 15
Add RNA to each tube. Then add 15 ul of master mix to each tube.
Incubate 25C for 10 mins
37C for 120 minutes
85C for 5-10 seconds
4C forever
11/26/2012 C:\Docstoc\Working\pdf\bff6c6be-9db3-4b89-9254-5690ec7fc8a4.xlsx
Instructions:
Add your values to the gray boxes. Mix ingredients
in the yellow boxes. You will generate two master
mixes: 1 & 2. Afterwards, follow the directions on
the sheet.
FAQ
How much RNA do I add? Typically I try to run
10 ug. However, 0.5-1 ug also works fine with
superscript. For lower concentrations, I
recommend Sensiscript (Invitrogen).
11/26/2012 C:\Docstoc\Working\pdf\bff6c6be-9db3-4b89-9254-5690ec7fc8a4.xlsx
Project: insert project name/lab book # here
Joshua Gray's Superscript III Protocol
Enter your Numbers Here:
Number samples 1
RT Reaction Volume 20 (usually 20, enough for 20 PCRs)
RNA volume 10 (up to 10 in a 20uL reaction)
Per reaction Master mix I Stock
Per Reaction
Random hexamers 0.5 0.55 500 ng/uL
dNTP mix 1 1.1 10 mM
water 0.5 0.55
Add 2 ul of Mix 1 to sample and heat to 65 for 5 min and chill on ice, then centrifuge
Per reaction Master Mix 2
5X First-Strand buffer 4 4.4
0.1M DTT 2 2.2
Rnasin 1 1.1
Superscript III 1 1.1
Add 8 ul of Mix 2 to sample
Incubate at 25C for 5 min
Incubate at 50C for 30-60 min
Incubate at 70C for 15 min
* Afterwards, dilute your cDNA 1:10. This prevents pipetting error when running your PCR. See the next pa
11/26/2012 C:\Docstoc\Working\pdf\bff6c6be-9db3-4b89-9254-5690ec7fc8a4.xlsx
Instructions:
Add your values to the gray boxes. Mix ingredients
in the yellow boxes. You will generate two master
mixes: 1 & 2. Afterwards, follow the directions on
the sheet.
FAQ
How much RNA do I add? Typically I try to run
10 ug. However, 0.5-1 ug also works fine with
superscript. For lower concentrations, I
recommend Sensiscript (Invitrogen).
What if my RNAs are different concentrations?
Dilute all of your RNA to the same concentration. It
makes life easier if you have to ever make more
RNA.
Why use Superscript over MMLV? I use
superscript for any work with animals. It gives
better results, but it is much more expensive.
MMLV works fine for cell lines.
Why do I dilute my cDNA 1:10 after the
reaction? Pipetting error is huge when you pipet
only 1 uL. Dilute your cDNA in the original tube
after the reaction, and then run 10 uL in the PCR
reactions.
our PCR. See the next page.
11/26/2012 C:\Docstoc\Working\pdf\bff6c6be-9db3-4b89-9254-5690ec7fc8a4.xlsx
Project: insert project name/lab book # here
Joshua Gray's MMLV Reverse Transcriptase Protocol
MMLV RT Reaction Enter values below:
Number of RXNS 1
Sample Volume 10 (liquid volume added RNA, up to 10uL in a 20uL reaction)
Reaction Volume 20 (normally 20uL)
RNA Quantity 1 ug (usually 1ug. If smaller, do the superscript protocol instead)
This is what you are adding per reaction
Component Amount Volume (ul) Final Concentration
random hexamers 250 ng 1 12.5 ng/uL
total RNA 10 0.05 ug/ul
dNTP Mix 10 mM 1 0.5 mM
RT buffer 5X 4 1 x
RNAase inhibitor 40 u/ul 1 2 U/ul
Reverse Transcriptase 200 u/ul 1 10 U/ul
Water 2
MIX 1
Random hexamers 1.1
dNTP Mix 1.1
H20 2.2
Add 4 ul of Mix 1 to sample and heat to 65 for 5 min and chill on ice, then centrifuge
Mix 2
RT buffer 4.4
RNAase inhibitor 1.1
Reverse transcriptase 1.1
Add 6 uL of Mix 2 to sample and heat to 37 for 50-120 min
Heat inactivate by incubating at 70 for 15 minutes
11/26/2012 C:\Docstoc\Working\pdf\bff6c6be-9db3-4b89-9254-5690ec7fc8a4.xlsx
up to 10uL in a 20uL reaction)
the superscript protocol instead)
Instructions:
Add your values to the gray boxes. Mix
ingredients in the yellow boxes. You will generate
two master mixes: 1 & 2. Afterwards, follow the
directions on the sheet.
FAQ
How much RNA do I add? Typically I try to run
10 ug. However, 0.5-1 ug also works fine with
superscript. For lower concentrations, I
recommend Sensiscript (Invitrogen).
What if my RNAs are different concentrations?
Dilute all of your RNA to the same concentration. It
makes life easier if you have to ever make more
RNA.
Why use Superscript over MMLV? I use
superscript for any work with animals. It gives
better results, but it is much more expensive.
MMLV works fine for cell lines.
Why do I dilute my cDNA 1:10 after the
reaction? Pipetting error is huge when you pipet
only 1 uL. Dilute your cDNA in the original tube
after the reaction, and then run 10 uL in the PCR
reactions.
Project: insert project name/lab book # here
Instructions:
Add your values to the gra
in the yellow boxes. You w
mix. Afterwards, follow the
FAQ
SYBR Green Real-time Reaction: Enter values below: Can I run less than 25 uL
Number of Reactions 1 the reaction volume to wha
RT reaction volume 10 (usually 2uL, can go up to 10uL) everything will be adjusted
Reaction Volume 25 (usually 50uL) pipetting error.
Final Primer Concentration 300 nM (between 50-900nM, usually 300nM)
Primer Master Concentration 10 uM (enter concentration of the stock vial) Why do you add 10 uL o
like a lot! I add 10 uL of c
This is what you are adding per reaction already diluted it 1:10, to a
Component Volume (ul) Concentration
Final Pipetting 1 uL is not reprod
Why do you choose 300
Water 1.00
concentration?
Primer Forward 0.75 300 nM using 50-900 nM. Rather
Primer Reverse 0.75 300 nM throw away primers that d
SYBR Master Mix 12.50 don't have to think about in
conditions for each primer
Master Mix primers is cheaper than op
Component Final
Volume (ul) Concentration
Water 1.10
Primer Forward 0.83
Primer Reverse 0.83
SYBR Master Mix 13.75
Steps:
1. Prepare the Master Mix, as above, and store the mixture on ice.
2. Add the RT reaction to each well.
3. Add this volume of SYBR mix to each well: 15 uL
4. Place optical caps or cover sheet over each of the tubes and run the reaction.
Instructions:
Add your values to the gray boxes. Mix ingredients
in the yellow boxes. You will generate your master
mix. Afterwards, follow the directions on the sheet.
Can I run less than 25 uL? Sure. Just change
the reaction volume to whatever you want, and
everything will be adjusted. I stay at 25 uL to avoid
pipetting error.
Why do you add 10 uL of cDNA? That seems
I add 10 uL of cDNA because I have
already diluted it 1:10, to avoid pipetting error.
Pipetting 1 uL is not reproducible.
Why do you choose 300 nM as the primer
concentration? Applied biosystems recommends
900 nM. Rather than optimize primers, I
throw away primers that don't work at 300 nM, so I
don't have to think about individual primer
conditions for each primer set! Ordering new
primers is cheaper than optimization!!!
Project: insert project name/lab book # here
Instructions:
Add your values to the gra
in the yellow boxes. You w
mix. Afterwards, follow the
FAQ
SYBR Green Real-time Reaction: Enter values below: Can I run less than 25 uL
Number of Reactions 138 the reaction volume to wha
RT reaction volume 10 (usually 2uL, can go up to 10uL) everything will be adjusted
Reaction Volume 25 (usually 50uL) pipetting error.
Final Primer Concentration 300 nM (between 50-900nM, usually 300nM)
Primer Master Concentration 10 uM (enter concentration of the stock vial) Why do you add 10 uL o
Dual-labeled probe Master 10 uM (enter concentration of the stock vial) like a lot! I add 10 uL of c
Dual-labeled probe final 100 nM (usually 100 nM) already diluted it 1:10, to a
Pipetting 1 uL is not reprod
This is what you are adding per reaction
Why do you choose 300
Component Final
Volume (ul) Concentration
concentration?
using 50-900 nM. Rather
Water 0.75 throw away primers that d
Primer Forward 0.75 300 nM don't have to think about in
Primer Reverse 0.75 300 nM conditions for each primer
Dual-labeled probe 0.25 100 nM primers is cheaper than op
TaqMan Master Mix 12.50
What is the dual
Master Mix a TaqMan-style PCR, you
Component Volume (ul) Concentration
Final binds in the middle. I use
(black hole quencher) labe
Water 113.85 purchased from IDT.
Primer Forward 113.85
Primer Reverse 113.85
Dual-labeled probe 37.95
TaqMan Master Mix 1897.50
Steps:
1. Prepare the Master Mix, as above, and store the mixture on ice.
2. Add the RT reaction to each well.
3. Add this volume of SYBR mix to each well: 15 uL
4. Place optical caps or cover sheet over each of the tubes and run the reaction.
Instructions:
Add your values to the gray boxes. Mix ingredients
in the yellow boxes. You will generate your master
mix. Afterwards, follow the directions on the sheet.
Can I run less than 25 uL? Sure. Just change
the reaction volume to whatever you want, and
everything will be adjusted. I stay at 25 uL to avoid
pipetting error.
Why do you add 10 uL of cDNA? That seems
I add 10 uL of cDNA because I have
already diluted it 1:10, to avoid pipetting error.
Pipetting 1 uL is not reproducible.
Why do you choose 300 nM as the primer
concentration? Applied biosystems recommends
900 nM. Rather than optimize primers, I
throw away primers that don't work at 300 nM, so I
don't have to think about individual primer
conditions for each primer set! Ordering new
primers is cheaper than optimization!!!
What is the dual-labeled probe? If you are doing
style PCR, you have a third primer that
binds in the middle. I use 5'-FAM and 3'-BHQ
(black hole quencher) labeled probes that can be
purchased from IDT.
