Koch Lab:Protocols/Dig-bio PCR: Difference between revisions
No edit summary |
|||
| Line 8: | Line 8: | ||
==A specific protocol, 4411 basepair tethering DNA== | ==A specific protocol, 4411 basepair tethering DNA== | ||
[[User:Skoch3|Steve]] would recommend starting with a PCR reaction that already works very well and produces a product close to the desired tether length. Then purchase dig and biotin primers with the exact same sequence and see if it works. A specific fragment commonly used by several labs in the single-molecule field is a PCR product resulting from PCR on the pRL574 plasmid from the [http://www.bact.wisc.edu/landick/ Landick Lab]. Here is a protocol for creating [[/pRL574 4411 PCR|dig / bio labeled 4.4 kb PCR product from plasmid pRL574]]. | [[User:Skoch3|Steve]] would recommend starting with a PCR reaction that already works very well and produces a product close to the desired tether length. Then purchase dig and biotin primers with the exact same sequence and see if it works. A specific fragment commonly used by several labs in the single-molecule field is a PCR product resulting from PCR on the pRL574 plasmid from the [http://www.bact.wisc.edu/landick/ Landick Lab]. Here is a protocol for creating [[/pRL574 4411 PCR|dig / bio labeled 4.4 kb PCR product from plasmid pRL574]]. | ||
[[Category:Protocol]] [[Category:DNA]] | |||
[[Category:Koch Lab Page]] | |||
Revision as of 19:43, 9 August 2007
Principle
A very easy way to make a batch of uniform DNA molecules labeled at one end with digoxigenin (dig) and the other end with biotin is to perform a PCR reaction using primers that have covalently attached dig or biotin. To visualize how this works, look at this image from Wikimedia Commons User:Madprime. At the bottom of the image, the PCR products which are accumulating show the primer oligonucleotides in red. At the end of many rounds of amplification, almost all fragments will have these primers, and if they are purchased with covalently attached biotin or dig (or other labels, such as fluorescein) then each identical fragment will have the desired labels at each end. (Note that the labels will actually be on opposite strands, which in some cases is important--the simple PCR method does not allow for labeling of the same strand at opposite ends.)
Some general thoughts--not rigorously tested
- Purchase primers with 5' (5-prime) modifications. Because the polymerase in the PCR reaction will work from the 3' end of the primer, it's likely that the 5' modifications will not hinder the reaction.
- Biotin is widely used and is an inexpensive modification. Dig is less common and patent-protected and tends to be fairly expensive. Fluorescein has also been used in single-molecule assays (with corresponding anti-fluorescein) and is cheaper than dig, but not as widely used.
- Steve endorses Alpha DNA, especially for complicated orders. Probably most companies can provide good biotin oligos, but dig oligos tend to be more problematic (perhaps due to restrictive liscensing from Roche). We have used 200 nanomol scale with OPC purification.
A specific protocol, 4411 basepair tethering DNA
Steve would recommend starting with a PCR reaction that already works very well and produces a product close to the desired tether length. Then purchase dig and biotin primers with the exact same sequence and see if it works. A specific fragment commonly used by several labs in the single-molecule field is a PCR product resulting from PCR on the pRL574 plasmid from the Landick Lab. Here is a protocol for creating dig / bio labeled 4.4 kb PCR product from plasmid pRL574.
