Adding 3' A overhang to a PCR product
|Author: S. Mohsen Hosseini, Hospital for Sick Children, To|
|Source: Protocol Online|
|Date Added: Mon Feb 02 2009|
|Date Modified: Mon Feb 02 2009|
|Abstract: Protocol for adding 3' A overhang to PCR products for TA cloning.|
ADDING 3' A OVERHANG TO A PCR PRODUCT
|Final Concentration||Vol (μl)|
|Purified PCR product||0.15 to 1.5 pmol||Varies*|
|dATP (10 mM)||0.2 mM||1|
|PCR Buffer with Mg (10x)||1x (1.5 mM MgCl2)||5|
|Taq DNA Polymerase (5 U/μl)||1U||0.2|
|ddH2O||to 50 μl|
* The A-addition reaction works best when a specific amount of the PCR product is used. The recommended amount is 10100 ng PCR product for each 100 bp length of the PCR product. This corresponds to 0.151.5 pmol PCR product (see table below).
PCR product size Amount of PCR product to use 100 bp 10100 ng 250 bp 25250 ng 1000 bp 1001000 ng
Based on protocols from Qiagen and Finnzymes.