Dear Kannan,

I think that you pretty much have the idea, but agree that it would be good to be clear on the details of what you will order.

So, if the 3' end of your sequence is ATCGAGTCAGGGTCCAA, say, and you wish to add an NdeI site CATATG, with the three random bases as ATA, then your "end of gene" will be:

5'.....ATCGAGTCAGGGTCCAA CATATG ATA 3'

Now, to get the sequence for your primer, you need first to _reverse_ this to:

5' ATA GATATC AACCTGGGACTGAGCTA....3'

then _complement_ it to:

5' TAT CATATG TTGGACCCTGACTCGAT 3'

That should do the trick.

As a rule of thumb, I would aim for slightly more than 16 nucleotides of complementary sequence in this case (20-25 would be better), and would always want the 3' base on the primer to be a G or a C (as this has three H-bonds to the complementary base) - two G/C is better. Basically, it is better to pay €1 more for a longer primer, than waste €10 of reagents and your time on a failed PCR!

When you calculate the Tm, you must of course take into account your non-complementary bases at the 5' end of the primer. These will affect the Tm, for sure.

As an aside: this method of cloning (i.e. trying to restriction digest your PCR product) is a recipe for failure and frustration. Sometimes it works quickly; sometimes it doesn't. I've seen many students waste huge amounts of time on this, and there is no glory in cloning these days.

If you must use restriction enzymes, I would recommend first cloning into a generic vector (e.g. by TA cloning): you can then sequence this before you clone, and restriction enzymes work much better on plasmids than PCR products.

Better still, use one of the restriction enzyme free methods of cloning that are available nowadays. You might use Ligation independent cloning, PIPE (http://www.ncbi.nlm.nih.gov/pubmed/18988020), MEGAWHOP (http://www.ncbi.nlm.nih.gov/pubmed/21601687), or one of several other methods: all of these remove the need to use restriction enzymes, and usually have a very high (i.e. >95% if the PCR product is correct) success rate. My laboratory and some colleagues have been using LIC for over 5 years (vectors and protocol kindly supplied by Opher Gileadi of SGC Oxford), and it has worked first time for almost every worker, including undergraduate students with no independent lab experience.

I hope that this helps,

Nicholas Harmer

You can refer to the NEB technical support. They provided a list to tell you how many nt should be add to the end. The overhang enhances the interaction between enzyme and DNA leading to increase the cleavage activity. For EcoR1, 1 nt overhang is enough. The number of nt overhang that sufficient to cleavage is depends on enzyme. Generally, 6 nts on either side of their recognition site to cleave is required.

As to the primer Tm, you should take the overhang into account due to they will also become the template to be binding for primer in next PCR cycle.

Dear Kannan,

I think that you pretty much have the idea, but agree that it would be good to be clear on the details of what you will order.

So, if the 3' end of your sequence is ATCGAGTCAGGGTCCAA, say, and you wish to add an NdeI site CATATG, with the three random bases as ATA, then your "end of gene" will be:

5'.....ATCGAGTCAGGGTCCAA CATATG ATA 3'

Now, to get the sequence for your primer, you need first to _reverse_ this to:

5' ATA GATATC AACCTGGGACTGAGCTA....3'

then _complement_ it to:

5' TAT CATATG TTGGACCCTGACTCGAT 3'

That should do the trick.

As a rule of thumb, I would aim for slightly more than 16 nucleotides of complementary sequence in this case (20-25 would be better), and would always want the 3' base on the primer to be a G or a C (as this has three H-bonds to the complementary base) - two G/C is better. Basically, it is better to pay €1 more for a longer primer, than waste €10 of reagents and your time on a failed PCR!

When you calculate the Tm, you must of course take into account your non-complementary bases at the 5' end of the primer. These will affect the Tm, for sure.

As an aside: this method of cloning (i.e. trying to restriction digest your PCR product) is a recipe for failure and frustration. Sometimes it works quickly; sometimes it doesn't. I've seen many students waste huge amounts of time on this, and there is no glory in cloning these days.

If you must use restriction enzymes, I would recommend first cloning into a generic vector (e.g. by TA cloning): you can then sequence this before you clone, and restriction enzymes work much better on plasmids than PCR products.

Better still, use one of the restriction enzyme free methods of cloning that are available nowadays. You might use Ligation independent cloning, PIPE (http://www.ncbi.nlm.nih.gov/pubmed/18988020), MEGAWHOP (http://www.ncbi.nlm.nih.gov/pubmed/21601687), or one of several other methods: all of these remove the need to use restriction enzymes, and usually have a very high (i.e. >95% if the PCR product is correct) success rate. My laboratory and some colleagues have been using LIC for over 5 years (vectors and protocol kindly supplied by Opher Gileadi of SGC Oxford), and it has worked first time for almost every worker, including undergraduate students with no independent lab experience.

I hope that this helps,

Nicholas Harmer

Well simply, go through this link, which helps you better to lear the basics:

http://www.bioinformatics.org/primerx/index.htm

In short, antiparallel is just the reverse of a sequence, say AAAAACCCC the anti parallel would be CCCCAAAA... but when you do reverse(&)complement then it become GGGGGTTTTT. You can play around this by sketching a short sequence of your DNA and add any restriction site and make a primer by yourself first, then just confirm it by any available free softwares.

Dear Venugopal,

in case of pwd primer are right. But for reverse primer its very simple add always restriction site on 5' of the oligo seq. ie.,if seq is 5-'ATGCATGCATGA-3' in +frame then first of all you should revrse complement of the seq (5'-TCATGCATGCAT-3') and then add restriction site at 5' of this oligo. if restriction sitewith overhang seq is OOO- XXXYYY, now the final primer seq you will order will be 5'-OOOXXXYYYTCATGCATGCAT-3'

In the first cycle your overhang and RE site don't bind to any complementary sequence. It is at the annealing step of 3rd cycle when the whole primer finds a full complementary sequence to bind. When your primers have over hang and RE site your first PCR product will be produced at the end of cycle 3. So according to me you can try a intermediate Tm with overhang and RE which can be standarized easily. Please consider hairpin structure while you design such primers.

Guys thank you all so much for your explanations! The concept is much clearer now.

Nicholas, your answer made things much simpler and clearer to me! Thank you.

First make reverse complementary sequence for the reverse primer and add the restriction site sequence to its 5 prime end. Your second part of ques, yes overall sequence will be considered for m purpose instead the annealing sequence only.

@Hitesh Kumar: Sir, if RE site have high GC content this will produce high Tm and as well as flanking seq also can increase Tm. therefore for the first cycle primer may fail to bind bcoz of high Tm and resulted into failed reaction. Am i right Sir ?

@ Avneesh Kumar. This can overcome through step down PCR , as you perform it for random amplification of cDNA ends (RACE). And high GC content may be a requisite for some oligonucletides, as in this case. Hope you got it.