10 x A-attachment mix is a reagent comprising anti-KOD DNA polymerase antibody specific to KOD 3'→5' exonuclease activity (proof-reading activity)(1), as well as Taq DNA polymerase, which exhibits terminal transferase activity. PCR products from KOD-Plus- [Code No. KOD-201] and KOD FX [Code No. KFX-101] possess blunt ends due to 3'→5' exonuclease activity of the KOD DNA polymerase. The 10 x A-attachment mix allows for PCR products to acquire overhanging dA at the 3'-ends. Products with 3'-dA overhangs can be directly cloned into arbitrary T-vectors using ligation reagents, such as Ligation high Ver.2 [Code No. LGK-201].
- The attachment reaction is completed in 10 minutes at 60ºC.
- The cloning efficiency of dA-attached PCR products is as great as PCR products from Taq DNA polymerase.
- Attachment of dA at the 3' - ends of blunt end PCR products from KOD -Plus- [Code No. KOD-201], KOD FX [Code No. KFX-101].
<The dA attached products can be cloned into a T vector using ligation reagents such as Ligation high Ver.2[Code No. LGK-201]>
Store at -20ºC
|10 x A-attachment mix||25 µl|
Example 1. TA cloning of a 500 bp-PCR product amplified with KOD- Plus-
One microliter of 10 x A-attachment mix was added to 9 µl of unpurified 500 bp-PCR product amplified by KOD-Plus- [Code No. KOD-201]. The reaction was incubated at 60ºC for 10 minutes. Subsequently, a solution (7.5 µl) containing 1.5 µl treated PCR products and 75 ng T-vector was mixed with 7.5 µl Ligation high Ver.2 [Code No. TAK-301] and incubated at 16ºC for 30 minutes. E. coli DH5a competent cells were then transformed using 10 µl of the ligation mixture, and were cultured on LB/Amp (X-gal) plates overnight at 37ºC. Blue and white colonies were quantified, and the insert DNA was confirmed by colony-directed PCR using eight white colonies as templates. As shown in Fig. 1, cloning efficiency was increased by treating PCR products with 10 x A-attachment mix. All eight white colonies were determined to possess the 500-bp inserts (data not shown).
Fig. 1. Comparison of TA cloning efficiency of PCR products from KOD -Plus-
- Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126: 762-8 (1999)