2nd primer for N-term tagged
2nd PCR primer is designed not to depend on ORF, the following primers can be used universally for 1st PCR products for N-terminal tags.
If the tags to be inserted are in the table below, there is no need to investigate the design and cycle. Four different primers are required and prepared.
Please note primer concentrations as shown in the figure below, since there are two different concentrations, 10 μM and 100 nM.
If the tags to be inserted are in the table below, there is no need to investigate the design and cycle. Four different primers are required and prepared.
Please note primer concentrations as shown in the figure below, since there are two different concentrations, 10 μM and 100 nM.
Forward Primer NF2 ( 10 μM )
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_NF2 | CCCGCGAAATTAATACGACTCACTATAG |
Forward Primer NF1 ( 100 nM )
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_no-tag_NF1 | CCCGCGAAAT TAATACGACT CACTATAG |
| 2nd_His_NF1 | CGACTCACTA TAGGGCTCAC CTATCTCTCT ACACAAAACA TTTCCCTACA TACAACTTTC AACTTCCTAT TATGCATCAT CATCATCATC ATCTCCAGCA GGGAGGTACTA TG |
| 2nd_FLAG_NF1 | CGACTCACTA TAGGGCTCAC CTATCTCTCT ACACAAAACA TTTCCCTACA TACAACTTTC AACTTCCTAT TATGGACTAC AAGGATGACG ATGACAAGCT CCAGCAGGGA GGTACTATG |
| 2nd_HA_NF1 | CGACTCACTA TAGGGCTCAC CTATCTCTCT ACACAAAACA TTTCCCTACA TACAACTTTC AACTTCCTAT TATGTATCCT TATGACGTGC CTGACTATGC CCTCCAGCAG GGAGGTACTA TG |
| 2nd_strepⅡ_NF1 | CGACTCACTA TAGGGCTCAC CTATCTCTCT ACACAAAACA TTTCCCTACA TACAACTTTC AACTTCCTAT TATGTGGTCC CACCCTCAAT TCGAGAAGCT CCAGCAGGGA GGTACTATG |
| 2nd_XXX-tag_NF1* | CGACTCACTA TAGGGCTCAC CTATCTCTCTA CACAAAACAT TTCCCTACAT ACAACTTTCA ACTTCCTATT ATG NNN—NNN CTCCAGCAGG GAGGTACTAT G |
＊You can use other tag to replace the NNN-NNN part of 2nd_XXX_tag_NF1 to arbitrary tag.
Reverse Primer NR1 ( 100 nM )
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_NR1 | CCCTCGAAGGATCAGGCCCTTATGGCCGGATCCAA |
Reverse Primer NCR2 ( 10 μM ) : same as C-term tagged
| Primer name | Primer sequence 5′ to 3′ |
| 2nd_NCR2 | GGCCCCCCCTCGAAGG |