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realtime calculator (Excel)

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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
4/22/2012                                 C:\Docstoc\Working\pdf\e89319ab-6904-4d4d-a0a1-f6ae76ede11e.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
4/22/2012                                C:\Docstoc\Working\pdf\e89319ab-6904-4d4d-a0a1-f6ae76ede11e.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).
4/22/2012                                 C:\Docstoc\Working\pdf\e89319ab-6904-4d4d-a0a1-f6ae76ede11e.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
           4/22/2012                                  C:\Docstoc\Working\pdf\e89319ab-6904-4d4d-a0a1-f6ae76ede11e.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.
4/22/2012                                 C:\Docstoc\Working\pdf\e89319ab-6904-4d4d-a0a1-f6ae76ede11e.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
            4/22/2012                                  C:\Docstoc\Working\pdf\e89319ab-6904-4d4d-a0a1-f6ae76ede11e.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.

				
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