Products for DNA end modifications

Phosphorylation
Vectors and inserts digested by restriction enzymes contain the necessary terminal modifications (5 ́ phosphate and 3 ́ hydroxyl), while ends created by PCR may not. Typical amplification by PCR does not use phosphorylated primers. In this case, the 5 ́ ends of the amplicon are non-phosphorylated and need to be treated by a kinase, such as T4 Polynucleotide Kinase, to introduce the 5 ́ phosphate.

 

DNA end modifications
Cat. no. Product Size Special offer
M0201S T4 Polynucleotide Kinase, 10 000 units/ml 500 units 605,-
M0201L T4 Polynucleotide Kinase, 10 000 units/ml 2500 units 2 417,-

Dephosphorylation
Dephosphorylation is a common step in traditional cloning to ensure the vector does not recircularize during ligation. If a vector is linearized by a single restriction enzyme or has been cut with two enzymes with compatible ends, use of a phosphatase to remove the 5 ́ phosphate reduces the occurrence of vector re-closure by intramolecular ligation and thereby reduces the background during subsequent transformation.

Quick CIP is a heat-labile version of calf intestinal alkaline phosphatase (CIP). It gives you faster reaction setup (no supplemental additives like zinc required) and shorter incubation time, flexible reaction conditions (active in any restriction enzyme buffer, no clean-up required), and less enzyme required (high specific activity).

Cat. no.  Product Size Special offer
M0525S Quick CIP, 5000 units/ml 1000 units 979,-
M0525L Quick CIP, 5000 units/ml 5000 units 4 687,-

Blunting/End-repair
Blunting is a process by which the single-stranded overhang created by a restriction digest is either “filled in”, by adding nucleotides on the complementary strand using the overhang as a template for polymerization, or by “chewing back” the overhang, using an exonuclease activity. Vectors and inserts are often “blunted” to allow non-compatible ends to be joined.

DNA polymerases, such as the Klenow Fragment of DNA Polymerase I and T4 DNA Polymerase, included in the Quick Blunting Kit from NEB, are often used to fill in (5 ́→3 ́) and chew back (3 ́→5 ́).

Cat. no.  Product Size Special offer
E1201S Quick Blunting Kit 20 reactions 896,-
E1201L Quick Blunting Kit 100 reactions 3 583,-
The offers are listed excl. VAT and shipping charges, and is valid until 31.12.2021. Promotional code #BN3-21 must be referenced when ordering to receive the special prices