Q10.  IS IT POSSIBLE TO DIRECTLY SUBJECT CELL LYSATES OBTAINED BY

                       EITHER HEAT TREATMENT (98 for 2 minutes) OR PROTEASE

                       DIGESTION FROM EITHER MAMMALIAN CELLS OR E. coli CELLS

                       TO LA PCR AMPLIFICATION?

In the case of E. coli, it is possible. Lysates of heat-treatment-only can generate DNA

fragments in length around 10 kbp.

In the case of human cells (cultured), the extent of purification significantly affects the

length of extension in PCR amplification. Heat treatment alone has a limit of several

hundred bases, while double treatment with heat and protease may exceed 1 to 2 kbp.

For longer length amplifications, it is recommended to use purified DNA prepared with

our standard protocol described in the Chapter 2.

LA PCR amplification using E. coli cells prepared with heat treatment as a

template with the LA PCR Kit Ver. 2.1

When amplified with the LA PCR Kit Ver. 2.1, a significant number of fragments up to

10 kbp at least can be generated.

Reactions were conducted under optimal conditions following the directions provided

with LA PCR Kit Ver. 2.1: An E. coli cell culture (37 overnight in L-broth) was treated at

98 for 2 minutes and 2 used as a template in a 50 LA PCR reaction.

Applied volume:

6

1% Agarose gel

electrophoresis