7 replies to this topic

[arvind]

hi

i had problem in amplifying 1.6 KB gene from genomic DNA of filarial parasite.

1.i tried diffent annealing temp
2.different template conc
3.Mgcl2 conc

but iam getting a prod size of 483 bp instead of 1.6 KB.
my primer blast gives me the exact size of 1.6 kb itself.

my protocol

95 C 5min, 95 C 1 min, 56 1 min, 72 1 min, 30 cycles , 72 10 min

what might be my problem and what shall i do to get maxamplification.

i isolated the genomic DNA also. it's conc is 2 ug per ul.

thank you

[vetticus3]

try using longer times for the annealing temps.  Taq uses about a min per Kb, check the protocol for the enzyme you're using.

[leahf]

hi
how conserved is that region - perhaps you  have a mutant strain?
can you get a different template of this parasite?
if money no objective, maybe order a different set of primers and see what you get...

[arvind]

vetticus3, on Feb 7 2005, 12:19 AM, said:

try using longer times for the annealing temps.  Taq uses about a min per Kb, check the protocol for the enzyme you're using.

hi

i tried 2 min annealing as suggested here before but i get only the same prod size and also some non specific bands

thank you

[arvind]

arvind, on Feb 7 2005, 12:36 AM, said:

vetticus3, on Feb 7 2005, 12:19 AM, said:

try using longer times for the annealing temps.  Taq uses about a min per Kb, check the protocol for the enzyme you're using.

hi

i tried 2 min annealing as suggested here before but i get only the same prod size and also some non specific bands

thank you

hi

i collected Mf from infected jirds and pooled them. i used this to isolate genomic DNA from mf.

intially i got the same problem so i ordered for new set of primers but the results are same.

i doubt that my genomic preparation is not good. if so i should not get any prod.

iam confused.
people say that it may be the contamination of the plasmid. but i dont get a false band always.

help me out

[leahf]

we are trying but its hard...

1. to make sure your genomic preparation is good try amplifing a different known gene of this parasite (if there is any)
2. how about sequencing the product you got - to see if it is a part of your gene or contamination?

about the new primer set - where the new primers in the same region of the old ones? i would try primers located as far away from the originals as possible, even if it means amplifing only a small part of the gene - in case part of the gene in your strain is different or missing.

3. are the flanking areas of the gene known? if so, maybe try targeting your primers to that area...

[arvind]

leahf, on Feb 7 2005, 06:24 AM, said:

we are trying but its hard...

1. to make sure your genomic preparation is good try amplifing a different known gene of this parasite (if there is any)
2. how about sequencing the product you got - to see if it is a part of your gene or contamination?

about the new primer set - where the new primers in the same region of the old ones? i would try primers located as far away from the originals as possible, even if it means amplifing only a small part of the gene - in case part of the gene in your strain is different or missing.

3. are the flanking areas of the gene known? if so, maybe try targeting your primers to that area...

hi
though the primers are new but the are targetting the same the gene.

i will try for new primers but i like to have it  as a lost choice.

i will sequence that prod to know what really is that.

if you suggest me better protocols than sambrook.. genomic DNA extarction i will try them atlast.

thank you.

[badcell]

Hi. Some suggestions: I would increase the extension time, not the annealing time, to 2 min. How much DNA are you using as a template? Do not use more than 50 ng, it would be easier to amplify nonoptimal primer binding sites if you have excess template. Also, as someone suggested before, try to amplify another gene from your parasite to make sure you got the right DNA and it's in good condition. You can also try touch-down PCR: amplify the fist 5-10 cycles at a higher, more-stringent annealing temperature (58-60ºC) so as to build up a backup of the right product, and then do the next 20-30 cycles at your normal annealing temp. Hope some of this can be of any help!