PCR - PCR CONTROLS AND HOW TO DETECT POLYMORPHISM (Apr/15/2008 )
How do you decide the controls for pcr method if I am not using kit?
How to detect polymorphism?
This is the gene that I need to figure out. can someone help mr please?
5'
301 gtcagcccca tggtggtggc tggggacagc cacatggtgg tggaggctgg ggtcaaggtg
361 gtagccacag tcagtggaac aagcccagta agccaaaaac caacaygaag catgtggcag
421 gagctgctgc agctggagca gtggtagggg gccttggtgg ctacatgctg ggaagtgcca
481 tgagcaggcc tcttatacat tttggcaatg actatgagga ccgttactat cgtgaaaaca3'
- primers or probes
-A list of enzymes
-how to detect the polymorphisms
-Controls
-The concentrations and formulations of gels
-The concentrations and components of enzymatic reactions
-how the results of assay would appear including
controls, a homozygous individual and a heterozygote.
How to detect polymorphism?
This is the gene that I need to figure out. can someone help mr please?
5'
301 gtcagcccca tggtggtggc tggggacagc cacatggtgg tggaggctgg ggtcaaggtg
361 gtagccacag tcagtggaac aagcccagta agccaaaaac caacaygaag catgtggcag
421 gagctgctgc agctggagca gtggtagggg gccttggtgg ctacatgctg ggaagtgcca
481 tgagcaggcc tcttatacat tttggcaatg actatgagga ccgttactat cgtgaaaaca3'
- primers or probes
-A list of enzymes
-how to detect the polymorphisms
-Controls
-The concentrations and formulations of gels
-The concentrations and components of enzymatic reactions
-how the results of assay would appear including
controls, a homozygous individual and a heterozygote.
Sounds like another homework question. What ideas have you had so far?
I enter this gene sequence and got the primes. but rest of the stuff I am not sure.
How to detect polymorphism?
This is the gene that I need to figure out. can someone help mr please?
5'
301 gtcagcccca tggtggtggc tggggacagc cacatggtgg tggaggctgg ggtcaaggtg
361 gtagccacag tcagtggaac aagcccagta agccaaaaac caacaygaag catgtggcag
421 gagctgctgc agctggagca gtggtagggg gccttggtgg ctacatgctg ggaagtgcca
481 tgagcaggcc tcttatacat tttggcaatg actatgagga ccgttactat cgtgaaaaca3'
- primers or probes
-A list of enzymes
-how to detect the polymorphisms
-Controls
-The concentrations and formulations of gels
-The concentrations and components of enzymatic reactions
-how the results of assay would appear including
controls, a homozygous individual and a heterozygote.[/quote]
Sounds like another homework question. What ideas have you had so far?
[/quote]
Do you have any ideas about where the polymorphism(s) might be? If so, you'll need a primer that sits such that the 3'base is the polymorphism, or else you'll need to use something like a TaqMan or a Hybeacon probe.
If you don't know where any polymorphism(s) are, you'll have to sequence the gene.
I think the polymorphism in my sequence is "y" I found the primer around that "y"
But I am not sure how to detect it and how to figure out the enaymes? Is there any software? How do I show the reaction and Homozygote and heterozygote?
Do far what I did is this....
5’GTCAGCCCCA TGGTGGTGGC TGGGGACAGC CACATGGTGG TGGAGGCTGG GGTCAAGGTG
GTAGCCACAG TCAGTGGAAC AAGCCCAGTA AGCCAAAAAC CAACAYGAAG CATGTGGCAG
PROBE >>>>>>>>>>>>>>>>>>>>>>
-------
GAGCTGCTGC AGCTGGAGCA GTGGTAGGGG GCCTTGGTGG CTACATGCTG GGAAGTGCCA
TGAGCAGGCC TCTTATACAT TTTGGCAATG ACTATGAGGA CCGTTACTAT CGTGAAAACA 3’
ACTCCT GGCAATGATA GCACTT
LEFT PRIMER >>>>>>>>>>
ccca gtaa gcca aaaa ccaa Number of bases 20 TM 55.2 G/C ratio 45.0
RIGHT PRIMER <<<<<<<<<<<<<<
Ttcacgatagtaacggtcctca Number of bases 22 TM 58.6 G/C ratio 45.5
REAGENTS NEEDED
PCR reaction mix (25 µL reaction ) for 30 cycles each
REAGENTS FINAL VOLUME FINAL CONCENTRATION
Sterile water (pH 7.0) 20.7µL -
10x PCR Taq Buffer 2.5µL 1x (50 nM KCl + 10 mM Tris-HCl+1.5mM MgCL2)
dNTPs mix (25 mM each nucleotide) 0.2µL 200 µM (each nucleotide)
Primer mix (25 pmoles/µL each primer) 0.4µL 0.4 µM (each primer)
Taq DNA polymerase (enzyme) 0.2µL 1 Unit/25 µL
DNA template (100 ng/µL) 1.0µL 100 ng/25 µL
Gel concentrations-
1 g of agarose in 100 ml of 1X TAE or TBE buffer (gives a 1% gel). 6X gel loading buffer to sample
Ethidium bromide solution (10 µl per 100ml of buffer)
But I am not sure how to detect it and how to figure out the enaymes? Is there any software? How do I show the reaction and Homozygote and heterozygote?
Do far what I did is this....
5’GTCAGCCCCA TGGTGGTGGC TGGGGACAGC CACATGGTGG TGGAGGCTGG GGTCAAGGTG
GTAGCCACAG TCAGTGGAAC AAGCCCAGTA AGCCAAAAAC CAACAYGAAG CATGTGGCAG
PROBE >>>>>>>>>>>>>>>>>>>>>>
-------
GAGCTGCTGC AGCTGGAGCA GTGGTAGGGG GCCTTGGTGG CTACATGCTG GGAAGTGCCA
TGAGCAGGCC TCTTATACAT TTTGGCAATG ACTATGAGGA CCGTTACTAT CGTGAAAACA 3’
ACTCCT GGCAATGATA GCACTT
LEFT PRIMER >>>>>>>>>>
ccca gtaa gcca aaaa ccaa Number of bases 20 TM 55.2 G/C ratio 45.0
RIGHT PRIMER <<<<<<<<<<<<<<
Ttcacgatagtaacggtcctca Number of bases 22 TM 58.6 G/C ratio 45.5
REAGENTS NEEDED
PCR reaction mix (25 µL reaction ) for 30 cycles each
REAGENTS FINAL VOLUME FINAL CONCENTRATION
Sterile water (pH 7.0) 20.7µL -
10x PCR Taq Buffer 2.5µL 1x (50 nM KCl + 10 mM Tris-HCl+1.5mM MgCL2)
dNTPs mix (25 mM each nucleotide) 0.2µL 200 µM (each nucleotide)
Primer mix (25 pmoles/µL each primer) 0.4µL 0.4 µM (each primer)
Taq DNA polymerase (enzyme) 0.2µL 1 Unit/25 µL
DNA template (100 ng/µL) 1.0µL 100 ng/25 µL
Gel concentrations-
1 g of agarose in 100 ml of 1X TAE or TBE buffer (gives a 1% gel). 6X gel loading buffer to sample
Ethidium bromide solution (10 µl per 100ml of buffer)
OK, my suggestion would be to make two primers based on the forward primer. The first one will end '... ccaa CAT', and the other one will end '...ccaa CAC' . This covers the two alternatives for "Y". If you carry out two separate PCRs, then run the products on a gel, only one should produce an amplicon, assuming you have a homozygote. If you have heterozygote, both will amplify. Depending on which primer produces a PCR product, you'll know the sequence at the "Y" position.