Sequencing Primers Design

[Mega]

Hi!

I always wanted to know in which plasmid the 35S-GFP cassette (delivered with pGreenII) is in... Flanked by EcoRV on both sides.

So I chose to have it sequenced... Wanna know the NNN ...

35S-GFP sequence

NNNNNNNNGATATCGTACCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCAAGCTGATCCAAGGAGATATAACAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGATCTGTGCAGGAGAGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGGAATCGATTTCAAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCCACAACGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGCGAATTAATTCGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCGATATCNNNNNNNNNNNNNN

So my primers should be like:
3' NNGATATCGTACCCCTACTCCAAA 5' its complement

And 5'  CTAAAATCCAGATCGATATCNN 3'  exactly this

Right? (Not considering annealing temperature)


[Cathal Garvey (Phone)]

I'm not very knowledgeable about sequencing in practise, but I'd suggest stepping back from the edges a little. Sequencing reactions seem to need 50-100 nucleotides before they start producing reliable results.. take a look at some traces and you'll see what I mean, the "bumps" start out all over the place, only start lining up and becoming consistent after a span of rubbish.

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[jarlemag]

 

I suppose that's what you meant by "complement", but just to make sure: It's the *reverse complement* of the first sequence you'll want.

 

 -JP

[jarlemag]

Ah, nevermind, I didn't notice you switched the 5' and 3' around.

 

-JP

[Mega]

So my aporoach is correct, just 100 bp space needed?

thx

[SC]

Hi Mega,

 

In general, you shouldn't include "N" characters in the primer sequence unless you specifically want to account for an ambiguity.  In this case, moving your primer target out ~100b will also accomplish this.

 

Good luck! 

[Mega]

Yeah. The N won't be included, it was just to visualize where the primer is...