Genome walking - (Sep/02/2004 )
hi ,all
I want to ask that the Tm's of AP1 and AP2 are 59ˇăC and 71ˇăC,which is written in the USER MANUAL,but I get 51.1ˇăC and 57.9ˇăC in the software of PRIMER PREMIER 5.0. If I use different softwares I will get different TM values, but they are all lower than those in the USER MANUAL. So whether I should correspondly low the special primers(GSP)'s TM values( recommended >=67ˇăC), when I design them.How do you deal with them .Thank you!
what I said above is all about the Universal genomewalker kit
edss-
Yes, you will get different Tm estimates for primers using different software, as each uses a slightly different formula to calculate the Tm. I use the Genomewalker kit. I haven't figured out why the Tm of AP1 is so low when they say to design your genespecific primers with a Tm of 70... However, the best way to determine the annealing temperatures to use is trial and error. If you have access to a gradient thermocycler, I'd run a test on one or two samples using annealing temperatures from below 60 to 72.
I have found that an annealing temp of 65 works well with my samples and genespecific primers. I am using Expand polymerase systems from Roche, which preferentially extend at 68, and peltier-device thermocyclers from MJ Research. BTW, I work on grass. I have also found that I can get the human DNA supplied as a control with the kit to successfully amplify under conditions which won't amplify the grass DNA.
Hope this helps.
Turtle
Turtle :
Thanks for your useful help! I will try it as you said .
And I also have a question:
when you design the two nested primers GSP1(genespecific primer)and GSP2, what is the best distance from the GSP2 to the 5'end of my DNAsequence(when i want to upstream) ?And what's the best distance between the two nested primers ?
Thanks for a lot!!!
I don't think there is a "best", but there are points you should take into consideration.
For the distance between GSP2 and the known 5' end of your sequence, don't put GSP2 at the most 5' end of known sequence because if you get PCR product with GSP2 and have sequenced it, you have to check if the new sequence overlaps the 5' end of known sequence.
The distance between GSP1 and GSP2 is not important. They can even overlap.
mario2004:D
Thank you very much for your help!!!
edss
Mario is right, the distance isn't important. I generally try to design my GSPs far enough 3' that I can also squeeze in a sequencing primer 5' of te nested GSP for walking upstream. I direct sequence my products, and find that having the sequencing primer nested inside the GSP works better than just using the nested GSP as a sequencing primer.
Turtle
thank you for your help!
may i ask which software do you use to decide the TM values of the primers?
It's already March... but I just wanted to briefly mention about my experience with Seegene's DNA Walking SpeedUp Kit that kiwi mentioned. I had a friend who tried it to do BAC cloning and she recommended it to me.
I had a piece of known seuqence and wanted to find out the sequence in the unknown region.
It was very easy to use and I had about 1Kb for each walking. I think the advantage of using this kit is that you only get real products (save so much time).
However, you have to design 3 target specific primers(TSPs) for nested PCR and it's quite crucial to have good TSPs for good result.
If you do try, don't forget to complete 3 PCR reactions because I didn't get any bands until the 3rd PCR.
They have a small pack.. 10 reaction.. that's what I first tried.. 
[i hope you could elaborate on the TSPs... I am also interested of making use of seegene... how much did it cost? thanks!