I have set of primers in the lab with Ecor1 site without extra nucleotides either side.
cant i use them directly with exonuclease activity enzyme and go for cloning? will digestion be affected as there are no extra nucleotides?
or should i carry on with TA cloning? What is the main advantage in this case?
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#2
HomeBrew
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Posted 10 March 2010 - 04:30 AM
Hi shreelatha, welcome to the BioForum!
EcoRI cuts well close to the ends of DNA, unlike many other restriction enzymes (see here), so it might work in this case (note that the table I've linked to doesn't have an entry for digestion efficiency when there are no 5' bases, but EcoRI cuts at >90% when there is just one 5' base).
Many enzymes require several 5' bases to cut well, and some enzymes just don't work efficiently near the ends of DNA regardless. In cases like this, TA cloning of your PCR product is very helpful.
However, consider the cost and labor involved in this step, compared to the cost of just re-ordering the primers with four or five 5' bases added, and thus eliminating the TA step.
EcoRI cuts well close to the ends of DNA, unlike many other restriction enzymes (see here), so it might work in this case (note that the table I've linked to doesn't have an entry for digestion efficiency when there are no 5' bases, but EcoRI cuts at >90% when there is just one 5' base).
Many enzymes require several 5' bases to cut well, and some enzymes just don't work efficiently near the ends of DNA regardless. In cases like this, TA cloning of your PCR product is very helpful.
However, consider the cost and labor involved in this step, compared to the cost of just re-ordering the primers with four or five 5' bases added, and thus eliminating the TA step.
#3
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Posted 10 March 2010 - 06:12 PM
ok thank u very much. i will go head n order new set of primers.
HomeBrew, on Mar 10 2010, 09:30 PM, said:
Hi shreelatha, welcome to the BioForum!
EcoRI cuts well close to the ends of DNA, unlike many other restriction enzymes (see here), so it might work in this case (note that the table I've linked to doesn't have an entry for digestion efficiency when there are no 5' bases, but EcoRI cuts at >90% when there is just one 5' base).
Many enzymes require several 5' bases to cut well, and some enzymes just don't work efficiently near the ends of DNA regardless. In cases like this, TA cloning of your PCR product is very helpful.
However, consider the cost and labor involved in this step, compared to the cost of just re-ordering the primers with four or five 5' bases added, and thus eliminating the TA step.
EcoRI cuts well close to the ends of DNA, unlike many other restriction enzymes (see here), so it might work in this case (note that the table I've linked to doesn't have an entry for digestion efficiency when there are no 5' bases, but EcoRI cuts at >90% when there is just one 5' base).
Many enzymes require several 5' bases to cut well, and some enzymes just don't work efficiently near the ends of DNA regardless. In cases like this, TA cloning of your PCR product is very helpful.
However, consider the cost and labor involved in this step, compared to the cost of just re-ordering the primers with four or five 5' bases added, and thus eliminating the TA step.
