Hello, I amplified a gene that I plan of sequencing using TOPOcloning TA. I was wondering what changes could I make to reduce the amount of smearing (I attached the gel photo). I do have a long last elongation step at 72C of 30 mins. I want to make sure the As get added at the end of the products. The quality of the DNA is good; not great and the buffer I used is not new (I read that buffer could be the issue). I used about 200ng of template (poplar DNA; 42 Mb genome). Would reducing the last elongation step to 15min help significantly? Should I get new buffer? What other conditions can I change? Would TOPOcloning work good enough to get 10 positive colonies with these PCR products? Thanks!
Hi Estafania,
Your ladder looks like 1 Kb+ and your amplicon is around 650 bp(?). I would not extend this longer than a minute during final extension and be sure that your enzyme still has terminal transferase activity. The "A" addition is pretty much instantaneous. Also, 200 ng for a 42 MB genome is pretty high, I use 50 ng when cloning the human genome.
I've had better luck with KAN than AMP for TA cloning. For an insert this small, I'd expect that you'd get 50% of your colonies with an insert.
Good luck
Hi Estafania,
Your ladder looks like 1 Kb+ and your amplicon is around 650 bp(?). I would not extend this longer than a minute during final extension and be sure that your enzyme still has terminal transferase activity. The "A" addition is pretty much instantaneous. Also, 200 ng for a 42 MB genome is pretty high, I use 50 ng when cloning the human genome.
I've had better luck with KAN than AMP for TA cloning. For an insert this small, I'd expect that you'd get 50% of your colonies with an insert.
Good luck
hi
I agree with Shale . you should decrease your template amount between 20-50 ng and add 1ul DMSO in PCR work solutions, run for 35 cycle / final extension 72 c 5 min as enough .
good luck
Shale is correct.
Can I also just check whether the enzyme you're using has exonuclease activity (proof-reading). If it does, then it doesn't matter how long you run the last extension to get the 'A' tagged on because the enzyme would cleave it off.
If you're just using plain taq, then you're good to TOPO clone - just purify and add to vector/salt mix.
If you're using a proof-reading enzyme, then consider moving to plain taq (if your sequence is under 1000bp then you should be ok to use it to avoid random point mutations) or perform a 3' A overhang PCR - it is explained in the TOPO cloning manual.
Happy cloning!
Thanks everyone. I am using Econotaq, which supposedly adds a "C/G" at the end of the product, but people in my lab and publications claim that it can be used for Topo TA cloning. I guess it adds an A/T to some products.
I have not worked with Econotaq but did a quick google search. It seems it attaches a G and not an A to the end of a fragment, so I'd assume it is not useful for a T/A cloning and TOPO vectors.
I also think 200 ng for template is too much, the smear you see on the gel could be the actual genomic DNA you used as template. I usually go for 10ng as template in a 25-50 uL PCR reaction and for 35-40 cycles. Check your extension time, rule by thumb is 1 min for 1kb, so for a 650bp fragment I'd do about 40 seconds. Also, the last extension step of 30 min is also too long, go for 3-5 min, that is plenty.
The smear could be a sign that your starter DNA is dirty/degraded. May want to add some PVPP to your DNA extraction to reduce secondary compounds co-isolation that can interfere with PCR.
After the PCR do a column clean up or ExoSap step. I usually get a lot of wrong inserts if I don't clean it and do a direct cloning of the unpurified PCR products.
To find positive clones I usually do a quick colony PCR in12-25uL of 8-16 colonies per ligation event. This is faster than Plasmid isolation to find the four clones or so I want to have sequenced. For sequencing I then do a plasmid prep but only of the four positive clones.
Hi Natascha,
I read the same thing about Econotaq, but there are publication that claim using Econotaq for Topocloning and I used it before with the really long extension time. I think that the long extension time might help the polymerase to add the occasional A/T at the end instead of G/C. However, I decided to purchase Taq to improve the chances of having A/T inserted at the end of the product.
I just did PCR yesterday and I reduced my starting template to 35ng. I am currently doing 35 cycles. If the product is too light, I will increase it to 40 cycles. I also reduced my final extension time to 10 min; maybe I will reduce it to 5 min next time.
I always do colony PCR, so I will definitely be doing that!
Thanks!
@ Jai, a person should use carefulness in using software to modify image/pictures for publication. Some journals might reject some modified figures, especially those with intensive modification. See below:
https://www.councilscienceeditors.org/resource-library/editorial-policies/white-paper-on-publication-ethics/3-4-digital-images-and-misconduct/
(From the site) "It is recommended that authors report how image data were manipulated, even if the image manipulations are considered acceptable practice, or state that image data were not manipulated."
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