Cloning PCR Fragments
Zuo and Rabie describe a new one-step "Quick Assemble" method of precisely and simultaneously joining multiple DNA fragments followed by intramolecular ligation. This technique of plasmid assembly and circularization can be used to construct new plasmids with any promoter, resistance gene marker, restriction site, or tag.
The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.
from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16
Further reading: Assembly of DNA fragments into circular constructs
The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.
from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16
Further reading: Assembly of DNA fragments into circular constructs
Labels: cloning, PCR cloning
Reamplification of a PCR Band
The products of a polymerase chain reaction commonly contain a mixture of products due to the amplification of unwanted or non-specific PCR fragments. This results in a range of bands on a gel often referred to as a "ladder" or "smear". Only one of these bands represents the desired DNA fragment. It can be difficult to find the correct PCR parameters with which to obtain the correct band in a pure state while still maintaining yield. Attempting to purify the band by cloning all the reaction products and then probing the library for the correct DNA can be very tedious and time-consuming.
A simple "core sampling" procedure can be used to isolate the required fragment from a mixture of PCR products. This post-PCR procedure involves "coring" an agarose sample out of a gel and using the sample as template in another round of PCR. This is a good "work-around" to obtain unique bands from a background of a large number of unwanted DNA fragments. Having a visible band of the required size is recommended although it is still possible to attempt the technique on "right-sized" invisible bands if a visible band cannot be achieved.
The full protocol for this PCR technique is available on-line at Core-Sampling a PCR Band
Bibliography:
A simple "core sampling" procedure can be used to isolate the required fragment from a mixture of PCR products. This post-PCR procedure involves "coring" an agarose sample out of a gel and using the sample as template in another round of PCR. This is a good "work-around" to obtain unique bands from a background of a large number of unwanted DNA fragments. Having a visible band of the required size is recommended although it is still possible to attempt the technique on "right-sized" invisible bands if a visible band cannot be achieved.
The full protocol for this PCR technique is available on-line at Core-Sampling a PCR Band
Bibliography:
- PCR Troubleshooting: The Essential Guide
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Books
Labels: core sampling, PCR cloning, protocols, reamplification
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