Restriction Free Cloning

Construct Name
pPLASMID-Insert

Forward Primer
Plasmid annealing = 66°C     Target annealing = 56°C    Length = 41
AGCCCCAAAGCGCAACAAGCCCCCACCATATCCACCACCAC


Reverse Primer
Plasmid annealing = 60°C     Target annealing = 56°C    Length = 40
ACTCCCACTTGTCGTAGTTGGGTGGTGGTGGATATGGTGG

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
62bps 1224bps
861-879
18bps

2° PCR conditions
Extension Time ng of insert ng of plasmid
0:24 mins 14.9 14.7

New construct
ATGC = Original plasmid sequence. ATGC = Inserted sequence.

CXXGCGGTAG TACTGCAATT GXTAXGAAAG AAACCXGCTG CTGCTAAATT CGAACGCCAG CACATGGACA GCCCAGATCT GGGTACCGGT GGTGGXTCCG GTATTGAGGG ACGCGGGTCC GAGAATCTTT ATTTTCAGGG GCAGCAGCCT GGAAAAGTTC TTGGGGACCA AAGAAGGCCT AGTTTGCCCG CCCTGCATTT TATCAAAGGG GCAGGGAAGA GGGACTCATC GAGGCATGGG GGCCCACACT GCAATGTCTT TGTGGAACAC GAAGCCCTGC AGAGGCCAGT GGCATCTGAC TTTGAGCCCC AGGGTCTCAG CGAAGCAGCT CGATGGAACT CCAAGGAAAA CCTTCTTGCT GGGCCCAGTG AAAATGACCC CAACCTTTTT GTGGCACTCT ATGATTTTGT GGCCAGTGGA GATAACACTC TCAGCATCAC TAAAGGTGAA AAGCTCCGGG TCTTGGGTTA TAATCACAAT GGGGAATGGG CCGAAGCCCA AACGAAAAAT GGCCAAGGAT GGGTCCCAAG CAACTACATC ACCCCCGTCA ACAGTCTGGA GAAACACTCC TGGTACCATG GGCCTGTGTC CCGCAATGCC GCTGAGTATC TGCTGAGCAG CGGGATCAAT GGCAGCTTCT TGGTGCGTGA GAGTGAGAGC AGTCCTGGCC AGAGGTCCAT CTCGCTGAGA TACGAAGGGA GGGTGTACCA TTACAGGATC AACACTGCTT CTGATGGCAA GCTCTACGTC TCCTCCGAGA GCCGCTTCAA CACCCTGGCC GAGTTGGTTC ATCATCATTC AACGGTGGCC GACGGGCTCA TCACCACGCT CCATTATCCA GCCCCAAAGC GCAACAAGCC CCCACCATAT CCACCACCAC CCAACTACGA CAAGTGGGAG TAAGAGCTCC GTCGACAAGC TTGCGGCCGC ACTCGAGCAC CACCACCACC ACCACCACCA CTAATTGATT AATACCTAGG CTGCTXAACA AAGCCAGGCC TTGGCGCGCC TGCAGGCGAG CTCCGTCGAC AAGCTTGCGG CCGCACTCGA GCACCACCAC CACCACCACC ACCACTAATT GATTAATACC TAGGCTGCTA AACAAAGCCC GAAAGGAAGC TGAGTTGGCT GCTGCCACCG CTGAGCAATA ACTAGCATAA CCCCTTGGGG CCTCTAAACG GGTCTTGAGG GGTTTTTTGC TGAAAGGAGG AACTATATCC GGAT

Mar 29 2024



Plasmid markers and restriction enzymes:







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