I was just wondering if anyone can tell me how to put A overhangs on my insert for TA cloning? the reason i ask is that the fragment wont amplify so we are having to use an enzyme which creates blunt ends.
Thanks in advance
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#1
Kami23
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Posted 27 April 2009 - 04:58 AM
Hey all,
I was just wondering if anyone can tell me how to put A overhangs on my insert for TA cloning? the reason i ask is that the fragment wont amplify so we are having to use an enzyme which creates blunt ends.
Thanks in advance
I was just wondering if anyone can tell me how to put A overhangs on my insert for TA cloning? the reason i ask is that the fragment wont amplify so we are having to use an enzyme which creates blunt ends.
Thanks in advance
#2
Bomber
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Posted 27 April 2009 - 05:07 AM
Hi,
after the PCR you can purify your pcr product and then use a normal taq.
Also add fresh buffer and dNTP`s, Mgcl2 etc... .
Incubate samples for around 15minutes at 72°C. That's it.
Otherwise you can also use the taq directly after the pcr has finished but then you have to use the taq in a 1:10 ratio (regarding the Units), since otherwise the blunt enzyme will always cut away the A'.
Incubate as described above, done.
Good luck
after the PCR you can purify your pcr product and then use a normal taq.
Also add fresh buffer and dNTP`s, Mgcl2 etc... .
Incubate samples for around 15minutes at 72°C. That's it.
Otherwise you can also use the taq directly after the pcr has finished but then you have to use the taq in a 1:10 ratio (regarding the Units), since otherwise the blunt enzyme will always cut away the A'.
Incubate as described above, done.
Good luck
Edited by Bomber, 27 April 2009 - 05:15 AM.
#3
Kami23
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Posted 27 April 2009 - 05:34 AM
Bomber, on Apr 27 2009, 02:07 PM, said:
Hi,
after the PCR you can purify your pcr product and then use a normal taq.
Also add fresh buffer and dNTP`s, Mgcl2 etc... .
Incubate samples for around 15minutes at 72°C. That's it.
Otherwise you can also use the taq directly after the pcr has finished but then you have to use the taq in a 1:10 ratio (regarding the Units), since otherwise the blunt enzyme will always cut away the A'.
Incubate as described above, done.
Good luck
after the PCR you can purify your pcr product and then use a normal taq.
Also add fresh buffer and dNTP`s, Mgcl2 etc... .
Incubate samples for around 15minutes at 72°C. That's it.
Otherwise you can also use the taq directly after the pcr has finished but then you have to use the taq in a 1:10 ratio (regarding the Units), since otherwise the blunt enzyme will always cut away the A'.
Incubate as described above, done.
Good luck
thanks
just to clarify, would i have to use plain old taq? because we add pfu and this also amplifies in a blunt ended manner. Sorry if i sound a bit thick, its been a slow day
#4
Bomber
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Posted 27 April 2009 - 05:48 AM
Hi,
either you performa normal pcr with the pfu and then purify the product via column (if you have one specific product).
Then you use this eluate and add appropriate amount of Taq buffer, fresh dNTP's, MgCl2 (2mM for example) and the ususal amount of Taq (0,5 Units, whatever you take for regular PCR). Incubate for 15min at 72°C;
or you do not purify the PCR product an add the Taq directly to the Pfu PCR mix. Your last step in the PCR is probably 4°C hold;
you can simply add 10X more Taq units to the Pfu PCR (so if you used 0,5 units of Pfu, take at least 5U of Taq) incubate 15min 72°C. Purify the product or use it directly for TA-cloning.
Best regards
either you performa normal pcr with the pfu and then purify the product via column (if you have one specific product).
Then you use this eluate and add appropriate amount of Taq buffer, fresh dNTP's, MgCl2 (2mM for example) and the ususal amount of Taq (0,5 Units, whatever you take for regular PCR). Incubate for 15min at 72°C;
or you do not purify the PCR product an add the Taq directly to the Pfu PCR mix. Your last step in the PCR is probably 4°C hold;
you can simply add 10X more Taq units to the Pfu PCR (so if you used 0,5 units of Pfu, take at least 5U of Taq) incubate 15min 72°C. Purify the product or use it directly for TA-cloning.
Best regards
