2nd primer for C-term tagged
Since the 2nd PCR primer is designed not to depend on ORF, the following primers can be used universally for the products of 1st PCR for C-terminal tag.
If the tags to be inserted are in the table below, there is no need to consider the design and cycle. Three types of primers are required.
Please note that primer concentrations are not the same as shown in the figure below, two different concentrations, 10 μM and 100 nM.
If the tags to be inserted are in the table below, there is no need to consider the design and cycle. Three types of primers are required.
Please note that primer concentrations are not the same as shown in the figure below, two different concentrations, 10 μM and 100 nM.
Forward Primer CF1 ( 10 μM )
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_CF1 | CCCGCGAAAT TAATACGACT CACTATAGGG CTCACCTATC TCTCTACACA AAACATTTCC |
Forward Primer CR1 ( 100 nM )
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_His_CR1 | CCCTCGAAGG ATCAGGCCCT TATGGCCGGA TCCAAGAGCT CTTTTTTTTT TTTAATGATG ATGATGATGA TGAGTACCTC CCTGCTGG |
| 2nd_FLAG_CR1 | CCCTCGAAGG ATCAGGCCCT TATGGCCGGA TCCAAGAGCT CTTTTTTTTT TTTACTTGTC ATCGTCATCC TTGTAGTCAG TACCTCCCTG CTGG |
| 2nd_HA_CR1 | CCCTCGAAGG ATCAGGCCCT TATGGCCGGA TCCAAGAGCT CTTTTTTTTT TTTAGGCATA GTCAGGCACG TCATAAGGAT AAGTACCTCC CTGCTGG |
| 2nd_strepⅡ_CR1 | CCCTCGAAGG ATCAGGCCCT TATGGCCGGA TCCAAGAGCT CTTTTTTTTT TTTACTTCTC GAATTGAGGG TGGGACCAAG TACCTCCCTG CTGG |
| 2nd_XXX-tag_CR1* | CCCTCGAAGG ATCAGGCCCT TATGGCCGGA TCCAAGAGCT CTTTTTTTTT TTTA RRR—RRR AGTACCTCCC TGCTGG |
＊ You can change RRR—RRR at 2nd_XXX_tag_CR1 to use other tag
Reverse Primer NCR2 ( 10 μM ) : same for N-terminus tag
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_NCR2 | GGCCCCCCCTCGAAGG |