Lengthy primers - (Apr/10/2008 )

Hello people
I just want to have some opinion on how long a primer can be. Just to mention that i need to clone my fragment in mammalian expression plasmid. For that I will have to add ATG, Kozak and a few nucleotides (4) before the restriction site and of course restriction site. All this amounts to 16 nucleotides atleast (if i use XhoI site which has G at -3 position with respect to immediately following ATG so no need to add another codon which could make this region 19 nucleotide long) And now i have this clone as my template which is i guess the most tricky one. no matter which region i target of this clone, i face a problem of AT rich regions at one end and GC rich regions at the other end so primers dont match in their Tm.
Coming to the original question, i just want to know if i make my primer around 32-35 nucleotide long (with all 5' additions) would that work nicely?
Expert opinions are required

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32-35 nt is fine. That's not your biggest problem, or hurdle. That honour belongs to the fact you have AT- and GC-rich ends. If you can get your Tms close to each other, within, say, 4C of each other, the PCR should work. How bad is the AT and GC precentage for the two ends?

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I used 62 nt primer before in PCR for cloning and worked just fine.

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The AT and GC rich regions can be handled if i increase the length of my primer. I mean i have these regions with in that area which prohibit me to make less than 30 nt primer. I am pasting the 30 nt regions from 5' and 3' ends of the regions i need to amplify for you people to analyse
5' ATGGGTTGCT CTTTCTCTAT CTTCCTTCTT GCTTTACTTT.............
.........GGTCGCTA TCATCATGAT CATGTTTTCA GGGGTCGATG CC 3'

I have these primers for this region Forward AGTC CTCGAG ATGGGTTGCTCTTTCTC Tm = 50 (complementary region only)
Reverse AGTC AAGCTT TTA GGCATCGACCCCTG Tm = 52

Then I have this region
5' ctagagtggcggaatacatctggcctttatatccttacta..........
.........ggtcgctatcatcatgatcatgttttcaggggtcgatgcc 3'

Another region
5' atgcagggtaactgggccaaggtcgctatcatcatgatca........
......cctttggctgatgctgatgatatcacaggcagaagcataa 3'

Yet another region
5' agcacacacatcaccggtggctctgcggctcgtcaagccc.........
........cctttggctgatgctgatgatatcacaggcagaagcataa 3'

All these sequences are from same clone (a larger fragment cloned in a plasmid) and i need to amplify these regions for different functional studies. As I have already mentioned, I have to include all the regulatory sequences, ATG (where not present) Kozak, stop codon, restriction site, 4 nucleotides before RS

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We just made a 65 base primer and it worked fine.

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I wonder wat were the Tm of these primers. Did u use ur primers for overlap extension kind of PCR or just routine PCR amplification? By wat method u made ur primers? any software of manually like i have to make them?

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I am waiting for some more opinions

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Hi Muhammad,

In fact, I used them for overlap PCR. I was trying to introduce short sigalling peptide to my insert by PCR it was 72 nts long. I used two primers both were around 62 nts.
I used clone manager to synthezise them. Here is the protocol I used
98C-45" 98C-11" 45C-30" 72C-2' 72C-5' 35 cycles (fisrt round)
I had first to extract my band, since the PCR produced several bands due to nonspecific binding. I cut out the band that was approximately 1500 nucleotides (my band)
98C-45" 98C-11" 45C-30" 72C-2'15" 72C-5' 35 cycles (second round)
I used 2.5 uL of PCR product from the Step 1 PCR reactions as template in these reactions.
you might need to adjust your annealing temp according to your primers

Then, I extracted my band (~1600nt) because other bands were present when PCR was run on gel.
I did partial digestion for 15 min ( since I had more than one site in the insert)

after that, I did a linear ligation bweteen this PCR digested product and another product

Then, I PCRed the entire construct using 2.5 uL of ligation product as template using the last primer I used in the second PCR round and another antisense primer for the ligated fragment
gel extraction of my band
and finally ligation to my vector

I hope this will help

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thanks anwar for the info u have provided. this protocol of urs might help me at a later stage of my work as i intend to introduce ubiquitin to my insert after amplifying it. but at present i just have the confusion that whether my approximately 35 nt primers would work or not using this larger fragment (that is cloned into a plasmid and is 1758 bp long) as template. obviously very less chances, in fact approaching zero, of nonspecific binding. i just have the concern if they will actually bind to my template at the selected annealing temperature and will subsequently amplify it successfully

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its been quite some days that i posted this thread but it still awaits the concluding opinions. can anybody help me with this issue? im a little skeptic if my primers wud work or not. 35 nt primers................... wat can be the issues?

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