User:Anthony Salvagno/Notebook/Research/2011/02/24/New pALS Reverse Primer: Difference between revisions
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==Sequence== | ==Sequence== | ||
*5' - Digoxygenin - TTCGCTCCAAGCTGGGCTGTGTG | *5' - Digoxygenin - TTCGCTCCAAGCTGGGCTGTGTG | ||
==New Unzipping Adapter== | |||
I also put in for a new unzipping bottom adapter. The previous oligo was this:<br /> | |||
<pre> | |||
Oligo #2: Bottom adapter 1a - Biotin | |||
Sequence 5' to 3': GAGCGGATXACTATACTACATTAGAATTCAGAC | |||
Modification: X = biotin-dT | |||
</pre><br /> | |||
So I am now replacing the X with a T and putting the biotin on the 5' end. If there is a nick, this should help with unzipping. I don't think there can not be a nick with the biotin here so we shall see. | |||
[[Category:Ant Sequence]] | [[Category:Ant Sequence]] | ||
Latest revision as of 08:14, 24 February 2011
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Reason
I'm constantly getting two PCR products and the reason is because there is a sequence that matches my previous reverse primer by 67%. So I've checked through my pALS sequence and came up with another sequence that has a decent melting temp and has less matches. Here the next best match is 60% which is good enough (the forward primer has a 60% match too and that yields one product so this should work).
Sequence
- 5' - Digoxygenin - TTCGCTCCAAGCTGGGCTGTGTG
New Unzipping Adapter
I also put in for a new unzipping bottom adapter. The previous oligo was this:
Oligo #2: Bottom adapter 1a - Biotin Sequence 5' to 3': GAGCGGATXACTATACTACATTAGAATTCAGAC Modification: X = biotin-dT
So I am now replacing the X with a T and putting the biotin on the 5' end. If there is a nick, this should help with unzipping. I don't think there can not be a nick with the biotin here so we shall see.
