help TA cloning!

szeitlin at biomail.ucsd.edu szeitlin at biomail.ucsd.edu
Wed Dec 18 18:07:59 EST 2002


Hi,

there are a few tricks to using TA cloning methods that you should know. The control they give 
you usually has the ideal bases next to the overhang that actually ligates- this can make a 
difference. Just because the positive control is working doesn't  mean your sample is weird, it's 
probably just less efficient. Or, it might be weird for other reasons (see #4 below).

Some tricks that might help you:

1- your insert is pretty big. Make sure you band-purify (i like the qiagen gel extraction spin 
columns) and then do an extra round just incubating your purified insert with ATP and taq to get 
the overhang on the insert. there should be instructions for doing this somewhere in the 
troubleshooting section of the protocol, but in reality everyone should probably do this step all 
the time. it makes a HUGE difference.

2- always do the blue-white screening and make sure your Xgal is very fresh. this will save you a 
lot of pain and suffering.

3- best to start with fresh vector directly from the freezer- the overhang can fall off if you've 
been freezing and thawing a lot or leaving it at room temp or on ice for too long.

4- try transforming into different bacteria- like SURE cells, something that will be stable and block 
any weird rearrangements or instability intrinsic to your sequence.

i hope those help you!

sam

manojmadhav at hotmail.com wrote:


> Hi All!
> i write this to seek your help for my  problems with pBADThioTOPO vector from
> invitrogen.

> the vector is 4.4kb and the insert am trying to insert is 2.4 kb. i ligated
> straight from PCR reaction mix(Taq amplification), as well as with gel purified
> PCR band. in the latter case i get only few colonies, while first one gives
> plenty.i got a lot of transformation after ligation, but all appeared to be of
> the size of vector to vector ligation. but further analyses showed that its not
> vector to vector ligation. there is also an extra low intensity(<5%) high
> molecular size band in my PCR product. but i wonder how a low quantity large
> size band can surpass a 
> high quantity(95%) small size band to liagte to vector! I have screened around
> 300 clones from foue ligation reactions done in different ways, but of no avail!

> i also tried to ligate the company-supplied control, which worked well as
> evidenced by blue-white selection. I simply dont understand why my insert is not
> getting ligated, while the control template and primers supplied by manufacturer
> but amplified by myself using the same conditions as with my isert is doing well.
> i wish someone would have something to share with me to save myself, and
> probably to others who have same problem..
> best regards
> Manoj N K
> Yale university

> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&begin=0





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