June 27, 2006 at 7:17 pm #5128
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 ?
June 27, 2006 at 9:48 pm #50647
what differs the primers from eachother and are they complementary to the DNA?
How do u combine the primers?
June 27, 2006 at 11:21 pm #50649
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
June 28, 2006 at 9:26 am #50685
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?
June 28, 2006 at 5:26 pm #50700
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! 😕
June 28, 2006 at 7:04 pm #50704
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
June 28, 2006 at 9:58 pm #50714
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 .
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