Koch Lab:Protocols/Dig-bio PCR/pRL574 4411 PCR

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(New page: ==Materials== ===Primers=== ====Forward Primer==== 5'-dig-GTAAAACGACGGCCAGTGAATTC (The Koch Lab calls this "pRL574-F853-dig") * Should hybridize to the forward strand starting at basepair ...)
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This is the specific protocol for producing 4,411 base pair PCR fragments having a biotin label on the 5' end of one strand and a digoxigenin (dig) label on the 5' end of the other strand.
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==Materials==
==Materials==
===Primers===
===Primers===
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====Primer Vendor====
====Primer Vendor====
[[User:Skoch3|Steve]] endorses [http://www.alphadna.com/index.html Alpha DNA] for oligonucleotides.  There are many other vendors--it's a good idea to go with a recommendation for a good digoxigenin-labeled oligo vendor.
[[User:Skoch3|Steve]] endorses [http://www.alphadna.com/index.html Alpha DNA] for oligonucleotides.  There are many other vendors--it's a good idea to go with a recommendation for a good digoxigenin-labeled oligo vendor.
 +
===Plasmid===
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Plasmid pRL574, developed by the [http://www.bact.wisc.edu/landick/ Landick Lab].  This plasmid was developed for E. coli RNA Polymerase transcription assays and has a promoter, an ORF for the beta subunit of RNAP, and a terminator.  For many single-molecule DNA stretching assays, these genetics are not important and use of this plasmid is for legacy reasons.
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===PCR Materials===
 +
====Standard PCR mixes and taq polymerase====
 +
====[http://www.stratagene.com/products/displayproduct.aspx?pid=419 Perfect Match] PCR enhancer from Strategene====
 +
We have used this in the past to reduce smearing (presumably variable product lengths) in the PCR reaction.  However, a gut feeling says that the reaction could be optimized without this expensive reagent, either by simply tweaking the reaction, or by using better primers.
 +
===Reaction clean-up===
 +
One of our favorites is [http://www1.qiagen.com/Products/DnaCleanup/GelPcrSiCleanupSystems/MinElutePCRPurificationKit.aspx?rp=1000131&rpg=0 PCR Cleanup Kits] from QIAGEN.
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==Reaction Mixture==
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{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse; width:710px" <!-- This line here formats your table for you.  Change the code to change the formatting of your table.-->
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| align="center" style="background:#f0f0f0;"|'''Component'''
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| align="center" style="background:#f0f0f0;"|'''Amount (µl) to add to 100 µl reaction'''
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<!-- Copy and paste one of the lines above to create a new column in the schedule table.  Alternatively, you can also delete lines to reduce the number of columns.-->
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|--
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| Nanopure Water
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| 70
 +
|--
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| 10X PCR Buffer, No Mg
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| 10
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|--
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| pRL574 miniprep, 1.5 ng / µl (in our case a 1:100 dilution of stock)
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| 1
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|--
 +
|25 mM complete dNTP mix (July 2004 Invitrogen)
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| 1
 +
|--
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| pRL574-F853-dig 10 µM stock
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|5
 +
|--
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|pRL574-R5263-bio 10 µM stock
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|5
 +
|--
 +
|50 mM MgCl2
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|6.5
 +
|--
 +
|Perfect Match (1U / µl)
 +
|1
 +
|--
 +
|Taq (5U/µl)
 +
|0.50
 +
<!-- To add another row to the table copy and paste everything from the |-- line to just above this line.-->
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|}

Revision as of 01:46, 5 April 2007

This is the specific protocol for producing 4,411 base pair PCR fragments having a biotin label on the 5' end of one strand and a digoxigenin (dig) label on the 5' end of the other strand.

Contents

Materials

Primers

Forward Primer

5'-dig-GTAAAACGACGGCCAGTGAATTC (The Koch Lab calls this "pRL574-F853-dig")

  • Should hybridize to the forward strand starting at basepair 853

Reverse Primer

5'-bio-GGAAACAGCTATGACCATGATTAC (The Koch Lab calls this "pRL574-R5263-bio")

  • Should hyrbridize to the reverse strand starting at basepair 5263

Control Primers

It is not too expensive to purchase non-labeled primers to use as controls in case the PCR reaction doesn't work. Also sometimes it is convenient to perform PCR with only one label.

Primer Vendor

Steve endorses Alpha DNA for oligonucleotides. There are many other vendors--it's a good idea to go with a recommendation for a good digoxigenin-labeled oligo vendor.

Plasmid

Plasmid pRL574, developed by the Landick Lab. This plasmid was developed for E. coli RNA Polymerase transcription assays and has a promoter, an ORF for the beta subunit of RNAP, and a terminator. For many single-molecule DNA stretching assays, these genetics are not important and use of this plasmid is for legacy reasons.

PCR Materials

Standard PCR mixes and taq polymerase

Perfect Match PCR enhancer from Strategene

We have used this in the past to reduce smearing (presumably variable product lengths) in the PCR reaction. However, a gut feeling says that the reaction could be optimized without this expensive reagent, either by simply tweaking the reaction, or by using better primers.

Reaction clean-up

One of our favorites is PCR Cleanup Kits from QIAGEN.

Reaction Mixture

Component Amount (µl) to add to 100 µl reaction
Nanopure Water 70
10X PCR Buffer, No Mg 10
pRL574 miniprep, 1.5 ng / µl (in our case a 1:100 dilution of stock) 1
25 mM complete dNTP mix (July 2004 Invitrogen) 1
pRL574-F853-dig 10 µM stock 5
pRL574-R5263-bio 10 µM stock 5
50 mM MgCl2 6.5
Perfect Match (1U / µl) 1
Taq (5U/µl) 0.50
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