Primer dilution problem in RAPD PCR

[richadeo]

Hi guys,
I am working on genetic diversity of plant species using RAPD technique. I have ordered random primers in lyophilized form. I need to dilute them before using in pCR mix. How to make dilution for making working solution?
Can anybody please help me out.

[megha]

hello,
i am also working on the same,,,,
primer must be provided with further necessary informations for dilution,,
otherwise you should visit on website for operon primer dilution..

megha
INDIA

[Priyanka]

Hi
Its very simple... There would be given necesary specifications on the vial indicating the micromolar /pico-molar concentration of the primer just use standard formulas.. (the dealer would have given you a written handout for that also use it...(they sometimes mention how mush water shud you take for what dilution...)) Just chill and calculate you`d be fine...
Cheers!!
Priyanka

[chovek69]

After redisolution of the lyophilised primer you should always check the conc. spectrophotometrically. Don't believe to the signs. I used to have alot of headaches in the past when blindly believing on the manufacterers' numbers.

[sweta]

Hi guys,
I am working on genetic diversity of plant species using RAPD technique. I have ordered random primers in lyophilized form. I need to dilute them before using in pCR mix. How to make dilution for making working solution?
Can anybody please help me out.

[megha]

dear,
you have to calculate the mM conc. of provided primer (may be it shud be mentioned with the prime) thn add required quantity of MQ to dilute them.
megha

[rifzak]

lyophilized primer has info of amount in nmole mentioned on its vial or on paper sheet. Resuspend it to 100 uM/L concentration. For example, for an amount of 28.5 nM of lyophilized primer (the amount is provided by the vendor) add 285 ul water or TE buffer and get 100 uM/L stock solution. To prepare working solution dilute it further in 10 uM/L (or 10 pm/ul) for that just take 10 ul stock solution and add to 90 ul water (1:10 dilution). From here u can get 1uM or 0.5uM or 0.2uM primer/50ul PCR reaction mix.
Formula to calculate the amount of 1x TE Buffer to add to produce a 100 µM (100 pmol/µL) stock solution is following
formula may be used:
Primer Amount (in nmol)/ 0.10 = Volume of 1x TE Buffer needed (in µL)
Example: 27 nmol of primer received; 27 / 0.10 = 270 µL; Adding 270 µL of 1x TE Buffer to the lyophilized primer will produce a 100 µM solution.
Add the calculated amount of sterile, ice-cold 1x TE Buffer to the lyophilized primer. Mix well until all of the primer is dissolved (The undissolved primer should be
easily visible as a dark blue pellet). Its up to you whether u make 1:10 dilution or directly 1:100 dilution.A 1:100 dilution with 1x TE Buffer solution will produce a 1.0 µM working solution (additional 1x TE Buffer is required). It is recommended to do aliquoting both the stock solution (100 µM) and the
working solutions (1.0 µM) into several sterile microcentrifuge tubes.