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Old 09-29-2019, 11:10 PM   #1
tom3432
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Default Can primers for PCR be duplicated?

Complete beginner question here, don't laugh: If I have some primers that have been synthesized, and I am close to running out of them, is there any way to duplicate them / amplify them / synthesize more of them myself? I don't know the sequences. Or is my only option to buy more? Thanks
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Old 09-30-2019, 12:48 PM   #2
cmbetts
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Yes, but with levels of difficulty ranging from trivial to very difficult. If they're for an NGS related application, there's a good chance they're just common primers you can pick from a catalogue or find the sequence by google. You could also potentially track down sequences if they've been used in a published paper.
If you've been using them for sequencing, they're probably in your FASTQ somewhere with the main issue being that it can be hard to derived their length, but can be guessed at by Tm analysis.
If all else fails, you can clone and sequence them, or make a ssDNA NGS library and sequence them if it's a big multiplex pool.
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Old 09-30-2019, 03:47 PM   #3
tom3432
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Quote:
Originally Posted by cmbetts View Post
Yes, but with levels of difficulty ranging from trivial to very difficult. If they're for an NGS related application, there's a good chance they're just common primers you can pick from a catalogue or find the sequence by google. You could also potentially track down sequences if they've been used in a published paper.
If you've been using them for sequencing, they're probably in your FASTQ somewhere with the main issue being that it can be hard to derived their length, but can be guessed at by Tm analysis.
If all else fails, you can clone and sequence them, or make a ssDNA NGS library and sequence them if it's a big multiplex pool.

Thanks for that. How would I get an idea of how much it would cost to sequence some unknown primers?
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Old 10-01-2019, 01:57 PM   #4
cmbetts
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Again, it's highly dependent on your situation.

You'll need to either buy a ssDNA library kit (usually a minimum 12rxn size) or put something homebrew together based on a published protocol. which means it could range from ~$1000USD having to buy a 12rxn kit for a single sample to ~$20 if you already have the components to homebrew a single rxn sitting in your lab freezer (likely if your lab works with miRNA or ancient DNA, unlikely otherwise).

Unless this is a very complex multiplex pool, you'll see the full library diversity in 100k reads (I don't trust demultiplexing results with fewer than that), but sequencing is by the lane, not the number of reads needed. It's been a few years since I last needed to outsource sequencing, but I'd guess you'd end up paying at least $1000USD to a service provider. If you (or a friend) has access to a sequencer or send out multiplexed pools regularly, you can spike it into the pool at ~<1% and get enough reads to do the needed analysis, in which case it's essentially free.

TL : DR $20-2000 depending on the reagents and infrastructure at hand, plus your time of course
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Old 10-01-2019, 08:19 PM   #5
tom3432
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Thank you very much for all your help, very interesting.
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Old 10-24-2019, 11:29 AM   #6
austinso
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I don't understand how you could have gotten to that point...? But anyway...

If you still have the amplicon, and some residual primer left over, then send them out for Sanger sequencing to identify the putative primer sites. Adjust length according to desired Tm and then get them synthesized (it is always cheaper to synthesize).

You could do this within a week.
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Old 10-24-2019, 01:56 PM   #7
radood
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You could try contacting the company that synthesized them originally (idt, sigma, etc..) provided you still have some identifier on the tube to ask if they will forward you the information. That being said, the label on the original tubes that come from sigma actually have the sequence if it is not too long. Good luck!
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