Using primers after DNA modification

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
molecular newbie
Garter
Garter
Posts: 14
Joined: Tue Jun 27, 2006 7:12 pm

Using primers after DNA modification

Post by molecular newbie » Tue Jun 27, 2006 7:17 pm

Im relatively new to this stuff but ive been doing research for the past month , working with 3 commerical primers and control/experimental DNA samples. I use the methylated specific PCR procedure and then gel electrophorisis for result. MY PROBLEM is that im not get consistent bands and even sometimes getting no bands at all. Does anyone have any suggestions or tips on how i might be mis-using the primers ?
thanx

oppox
Death Adder
Death Adder
Posts: 96
Joined: Sat May 13, 2006 11:20 am
Location: Sweden

Post by oppox » Tue Jun 27, 2006 9:48 pm

what differs the primers from eachother and are they complementary to the DNA?
How do u combine the primers?

molecular newbie
Garter
Garter
Posts: 14
Joined: Tue Jun 27, 2006 7:12 pm

Post by molecular newbie » Tue Jun 27, 2006 11:21 pm

they are 3 different primers that cleave 3 different genes from the DNA strand. They dont differ much , and they have enough complentary base pairs to cleave that specific gene. They are commercial primers and therfore they are already mixed togther , i just add them before i add my master mix. One problem i think im having is the annealing temp, or maybe they are just old primers, it could be the PCR machine , i just dont know. What do u suggest the correct amount of DNA , Primer, and wat type of TAQ would u suggest i use? im currently using Hot start Polymerase. Im going crazy cause i have a project to finish by the end of July i just need to start getting consistant results.

Thanx for your reply

oppox
Death Adder
Death Adder
Posts: 96
Joined: Sat May 13, 2006 11:20 am
Location: Sweden

Post by oppox » Wed Jun 28, 2006 9:26 am

ok. My PCR program is 95 degree (30 s) 50 deg (1 min) 68 deg (4 min) repeat 25 times then 68 deg for 10 min and last some cooling steps 60 deg (2 min) 55 deg (2min) 50 deg (2 min). With Taq polymerase (think its hot start). How does your gel look like? Do u get any bands at all?
Do u do any control reaction on something known to work, just to see if your mastermix and pcr machine works?

molecular newbie
Garter
Garter
Posts: 14
Joined: Tue Jun 27, 2006 7:12 pm

Post by molecular newbie » Wed Jun 28, 2006 5:26 pm

My gel is not the problem and i do use control DNA and i do get bands. I think my main problem is the primers not working correctly. When u make your master mix , how much DNA and primer do u put into each tube? and if for example your using 10 tubes wat is your Master mix consist of? and one last question , i do DNA isolation starting from scratching slides then using a kit to isolate the DNA , i then concentrate it using NaOAC , and ethanol , (not sure if u know the procedure) but after that i use another kit to modify the DNA so that i can tell the difference between Methylated and Unmethylated genes, so if you have done these procedures and have any tips or suggestions in order to improve my DNA yield or any good annealing temps , please let me know,
I really appreciate u responding thanx! :?

oppox
Death Adder
Death Adder
Posts: 96
Joined: Sat May 13, 2006 11:20 am
Location: Sweden

Post by oppox » Wed Jun 28, 2006 7:04 pm

oh. you are using a procedure that im not familiar with, why is it important that its methylated or not? If u want to get rid of methylated DNA u can always digest it with dpn1.

Whenever I have experienced any problem with a PCR run, something is wrong with the stuff im using. Wrong template usually.
I dont make master mixes (only did it ones to save time), im doing it after the novagen Taq polymerase pcr reciepe. You are doing (whats seems to me anyway) alot of steps before the actual PCR, is it possible the DNA is lost on the way?

But as I said when It didnt work for me I didnt have a correct template ( I was trying to see if I had the correct insert after cloning).
I dont know if any of this help, but ive been (am) in a simillar position so I know the feeling. good luck

molecular newbie
Garter
Garter
Posts: 14
Joined: Tue Jun 27, 2006 7:12 pm

Post by molecular newbie » Wed Jun 28, 2006 9:58 pm

yea i do a lot of steps before pcr and it does loose a lot of the DNA , but i think im just gonna go ahead and order new material cause i think your right , i need to start fresh. Thanx a lot for your time and help i appreciate it. ill update u later on how my works goin .



peace

Post Reply

Who is online

Users browsing this forum: No registered users and 1 guest