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