PCR-SOME USEFUL GUIDELINES - (Oct/17/2007 )
Hi,
I have alot of questions about PCR. My sample is fish tissue. Can some body VERY patient help?
1. which factor did u optimise first-primer/temp/concentration of DNA or other?
2.Is MgCl2 always used for PCR?
3.25 mM MgCl2-how long u can keep it, at what temperature, what bottle?
4.Primer-do you use readymade primer for your first PCR?
5.If not, how many bp length primer u start with?
6If u designed a primer did you determine the G-C content of it? How, through a programme?
7. which is best DNA extraction kit for a brginner?
8. How would you clean a tissue sample prior to DNA extraction?
9 How would u know tissue of a fish is free of microalgae and pathogens even after cleaning ?
10 How will u preserve tissues before DNA extraction?
11 do u take preserved tissue directly to the homogenization tube for D NA extraction procedure?
12 reaction buffer-u have to make it or is it readymade -which is better?
13 How long can keep a reaction buffer, at what temp, what bottle?
14. Reaction buffer-which is best?
15. how to make a 20 mM dNTP mix?
16. dNTPs-do they come in packets? as solid/powder?
17 Primer mix-how to make it? take out to a tube and mix it?
THANK YOU SO MUCH
I try to answer.
1) I will try to optimize the annealing temperature first, followed by concentration of DNA and then primer.
2) Mg2+ is required for polymerase activity.
3)-20oC. Check with the manufacturer's user manual
4)what do you mean by readymade primer?
5,6) at least 21bp for good annealing. BLAST it to make sure it's specific and check using Netprimer or Operon for the melting temperature, GC content and primer-dimers. 50% GC content is ideal.
7) I don't use any kit. If yours is zebrafish, ZFIN has good protocol.
10) put the tissue on ice. I usually extract immeidately.
Roche catalog has good guidelines for successful PCR. You can check it out.
1. which factor did u optimise first-primer/temp/concentration of DNA or other?
Temperature. I generally use about 100ng of DNA (genomic).
2.Is MgCl2 always used for PCR?
Yes. It is usually standard in the buffer. You can have buffer without MgCl2, and you add as much as is required for the PCR to work (sometimes more, sometimes less).
3.25 mM MgCl2-how long u can keep it, at what temperature, what bottle?
-20'C. This is usually in a small tube (1.5mL) and is standard in any polymerase pack (ie, polymerase, buffer, MgCl2, etc etc).
4.Primer-do you use readymade primer for your first PCR?
what does this mean? are you talking about T7, T3 or other primers like that?
5.If not, how many bp length primer u start with?
whatever is appropriate for the experiment. try pubmed or google and look up primers, and how they work.
6If u designed a primer did you determine the G-C content of it? How, through a programme?
primer3. once again, use google, and you will have lots of info on this. after you designit, blast it, check for secondary structure. seriously, google this topic for more info.
7. which is best DNA extraction kit for a brginner?
we used Qiagen. works well. or you could use trysol.
8. How would you clean a tissue sample prior to DNA extraction?
i don't. it get's cut, and frozen (dry ice + ethanol, then -70'C) until i'm ready to use it.
9 How would u know tissue of a fish is free of microalgae and pathogens even after cleaning ?
try google. i can't help you here. better still, try pubmed.
10 How will u preserve tissues before DNA extraction?
freeze.
11 do u take preserved tissue directly to the homogenization tube for D NA extraction procedure?
look at the protocols from the DNA extraction kit you are going to use. generally, you don't let the tissue thaw before grinding it up.
12 reaction buffer-u have to make it or is it readymade -which is better?
reaction buffer for what? PCR? never made it up. it comes with the polymerase.
13 How long can keep a reaction buffer, at what temp, what bottle?
are you talking about the polymerase+buffer+dNTP+MgCl2+DNA+water? or just the buffer?
the entire reaction should be used imideatly. buffer can be kept frozen (-20'C) for ages.
14. Reaction buffer-which is best?
check which polymerase you are going to be useing. different buffers for different polymerases.
15. how to make a 20 mM dNTP mix?
you mix dATP, dCTP, dGTP, and dTTP in the right concentration with water. this is not rocket science.
16. dNTPs-do they come in packets? as solid/powder?
try looking up fermentas + dNTP. yes, they come in a packet with all 4 dNTP. they are in a liquid.
17 Primer mix-how to make it? take out to a tube and mix it?
depends on how you order it. you can order it in liquid or in solid. if it's solid, i just add the right amount of sterilised water. once again, not rocket science.
THANK YOU VERY MUCH SHIRLEYLER AND VETTICUS . YOUR COMMENTS ARE INDEED HELPFUL . THANKS
Temperature. I generally use about 100ng of DNA (genomic).
2.Is MgCl2 always used for PCR?
Yes. It is usually standard in the buffer. You can have buffer without MgCl2, and you add as much as is required for the PCR to work (sometimes more, sometimes less).
3.25 mM MgCl2-how long u can keep it, at what temperature, what bottle?
-20'C. This is usually in a small tube (1.5mL) and is standard in any polymerase pack (ie, polymerase, buffer, MgCl2, etc etc).
4.Primer-do you use readymade primer for your first PCR?
what does this mean? are you talking about T7, T3 or other primers like that?
5.If not, how many bp length primer u start with?
whatever is appropriate for the experiment. try pubmed or google and look up primers, and how they work.
6If u designed a primer did you determine the G-C content of it? How, through a programme?
primer3. once again, use google, and you will have lots of info on this. after you designit, blast it, check for secondary structure. seriously, google this topic for more info.
7. which is best DNA extraction kit for a brginner?
we used Qiagen. works well. or you could use trysol.
8. How would you clean a tissue sample prior to DNA extraction?
i don't. it get's cut, and frozen (dry ice + ethanol, then -70'C) until i'm ready to use it.
9 How would u know tissue of a fish is free of microalgae and pathogens even after cleaning ?
try google. i can't help you here. better still, try pubmed.
10 How will u preserve tissues before DNA extraction?
freeze.
11 do u take preserved tissue directly to the homogenization tube for D NA extraction procedure?
look at the protocols from the DNA extraction kit you are going to use. generally, you don't let the tissue thaw before grinding it up.
12 reaction buffer-u have to make it or is it readymade -which is better?
reaction buffer for what? PCR? never made it up. it comes with the polymerase.
13 How long can keep a reaction buffer, at what temp, what bottle?
are you talking about the polymerase+buffer+dNTP+MgCl2+DNA+water? or just the buffer?
the entire reaction should be used imideatly. buffer can be kept frozen (-20'C) for ages.
14. Reaction buffer-which is best?
check which polymerase you are going to be useing. different buffers for different polymerases.
15. how to make a 20 mM dNTP mix?
you mix dATP, dCTP, dGTP, and dTTP in the right concentration with water. this is not rocket science.
16. dNTPs-do they come in packets? as solid/powder?
try looking up fermentas + dNTP. yes, they come in a packet with all 4 dNTP. they are in a liquid.
17 Primer mix-how to make it? take out to a tube and mix it?
depends on how you order it. you can order it in liquid or in solid. if it's solid, i just add the right amount of sterilised water. once again, not rocket science.