Can we use a PCR product as transcription template instead of pEU with a gene insert?
Yes, you can, using a method called “split primer PCR.” In this method, you can make a linear DNA template for transcription by using our Wheat Germ Premium Expression Kit-PCR along with two custom primers procured by the user. The split primer PCR consists of the following two steps: 1st PCR reaction: Use a custom sense primer containing a sequence derived from the cDNA of interest and a custom antisense primer derived from the plasmid containing the cDNA located about 1.6 knts downstream. 2nd PCR reaction: Use three primers, two sense and one antisense primers. The two sense primers are generic, made by adding the E01 enhancer and SP6 promoter to the 5′ end of the PCR product. The antisense primer is the same as that used in the 1st PCR reaction. Since the SP6 promoter sequence is split between the two generic primers and is not entirely contained in either one, expression of non-specific amplification products is eliminated and production of the target protein is maximized. Upstream
- Does the vector pMM1522 contain a transcription terminator downstream of the MCS? Or do I need to insert a transcription terminator downstream of my gene of interest?
- We would like to check PCR product sizes as part of our Quality Control. Where can I find insert sizes of the NIA Mouse Clone Set Clones?
- Can we use a PCR product as transcription template instead of pEU with a gene insert?