help TA cloning!

Wolfgang Schechinger hubahopp at gmx.de
Thu Dec 19 09:29:17 EST 2002


Hi guys, 

just a stupid question on Taq: It's known to add overhangs. So it would add
an overhang in *every* cycle, wouldn't it? Please find my mistake (today I
am as stupid as a pighead...)!

As alternative to TA cloning however, I'd suggest using primers that have
built in restriction site for your 2 favourite enzymes. Personally, I like
XhoI and HindIII, if possible and your sequence does not prevent their use.
Then you would need an appropriately modified vector of course (but only
need to do it once in a while). And if possible, use a proofreading enzyme.
Taq makes LOTS of errrors.

Work up procedure would be

- spin column purify your pcr product (to remove Taq, can be very nasty
sometimes afterwards when carried over, seems to survice even EtOH prec.)
- digest with DpnI (kills methylated template) and your 2 favourite enzymes
in one Rxn
- digest vector with the same 2 favourite enzymes and include some SAP(aso
in one RxN)
- spin column purify vector and insert, EtOH prec.
- ligate, transform, check. 

Usually works like a charm, except on mondays past full moon.

Wo

At 11:07 PM 2002/12/18 -0000, szeitlin at biomail.ucsd.edu wrote:
>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&be
gin=0
>
>
>
>
>
>http://biowww.net/mynews/tree.php?group_name=bionet.molbio.methds-reagnts&b
egin=0
>
>

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