I have a sticky-end insert but I need to clone it into ablunt-end plasmid, but I am not sure what strategy should I use. Is it better to use a polymerase and synthesize complemetary sequence to sticky-ends to create a blunt-end insert or to create sticky ends on plasmid. Could some of you also recommend the best enzyme to do that? I was thinking about Klenow fragment to create blunt ends but first I´d like to hear other opinions. Thanks.
Hi there,
I think it should be much easier to fill overhangs to generate blunt ends on the insert that creating compatible sticky ends on the plasmid. Klenow treatment is OK : it will fill 5' overhang in presence of dNTPs and cut off 3' overhang.
Hi there,
I think it should be much easier to fill overhangs to generate blunt ends on the insert that creating compatible sticky ends on the plasmid. Klenow treatment is OK : it will fill 5' overhang in presence of dNTPs and cut off 3' overhang.
1) Purify your PCR/ plasmid fragment to remove the unnecessary dNTPs/sequence
2) Klenov
3) Voila.
Make the following reaction with Pfu polymerase:
add your insert diluted in water, 5 ul of 10xPfu PCR buffer, 0,2 - 0,5 U of Pfu polymerase and 0,05 uM dNTPs mix, total volume of reaction 50 ul, 72C, 30 min.
clean up the insert and ligate to blunt ended plsmid
To make blunting ends, Quick Blunting Kit (New England Biolabs) is used to convert DNA with incompatible 5´ or 3´ overhangs to 5´ phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vector. If you not able afford this kit simply you go with sticky ends filling using Pfu Taq Polymerase or Klenow Enzyme.
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