pPLASMID-Insert
Forward Primer
Plasmid annealing = 63°C Target annealing = 57°C Length = 45
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TCCAGCCCCAAAGCGCAACAAGCCACCACCACCATATCCACCACC
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Reverse Primer
Plasmid annealing = 54°C Target annealing = 57°C Length = 44
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TTACTCCCACTTGTCGTAGTTGGGTGGTGGTGGATATGGTGGTG
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New construct
ATGC = Original plasmid sequence. ATGC = Inserted sequence.
Plasmid annealing = 63°C Target annealing = 57°C Length = 45
Reverse Primer
Plasmid annealing = 54°C Target annealing = 57°C Length = 44
The insert is fully synthesized by the primers. Use a 5 cycle PCR reaction (w/o any plasmid) to anneal and extend 5ng of each primer.
| 1° PCR Size | New Plasmid Size | Insert Sites | Insert Size |
| 70bps | 699bps |
|
27bps |
| 2° PCR conditions | ||
| Extension Time | ng of insert | ng of plasmid |
| 0:13 mins | 16.8 | 8.4 |
New construct
ATGC = Original plasmid sequence. ATGC = Inserted sequence.
AAAGAAACCG XTGCTGCTAA ATTCGAACGC CAGCACATGG ACAGCCCAGA TATGGGTACC GGTGGTGGCT CCGGTATTGA GGGACGCGGG TCCGAGAATC TTTATTTTCA GGGGCAGCAG CCTGGAAAAG TTCTTGGGGA CCAAAGAAGG CCTAGTTTGC CCGCCCTGCA TTTTATCAAA GGGGCAGGGA AGAGGGACTC ATCGAGGCAT GGGGGCCCAC ACTGCAATGT CTTTGTGGAA CACGAAGCCC TGCAGAGGCC AGTGGCATCT GACTTTGAGC CCCAGGGTXT CAGCGAAGCA GCTCGATGGA ACTCCAAGGA AAACCTTCTT GCTGGGCCCA GTGAAAATGA CCCCAACGCT GGGCCCAGTG AAAATGACCC CAACCTTTTT GTGGCACTCT ATGATTTTGT GGCCAGTGGA GATAACACTC TCAGCATCAC TAAAGGTGAA AAGCTCCGGG TCTTGGGTTA TAATCACCTG AGATACGAAG GGAGGGTGTA CCATTACAGG ATCAACACTG CTTCTGATGG CAAGCTCTAC GTCTCCTCCG AGAGCCGCTT CAACACCCTG GCCGAGTTGG TTCATCATCA TTCAACGGTG GCCGACGGGC TCATCACCAC GCTCCATTAT CCAGCCCCAA AGCGCAACAA GCCACCACCA CCATATCCAC CACCACCCAA CTACGACAAG TGGGAGTAA
Apr 12 2025
Plasmid markers and restriction enzymes:
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