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You can use Tm 51 degree celcius.
And an initial set 42 degree for 5 cycles and then 51 degree for 35 cycles.
In initial 5 cycles, extension time will be less, better to keep extension for 20-30 seconds only.
You go with this, i am sure you will get the amplification.
Good luck
Dr. Rupesh Kumar Singh
PhD (Plant Biotechnology)
Department of Biotechnology
Kumaun University, Nainital,
Uttarakhand, India-263139
Mob- +91-9911896375
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DrRupesh Kumar Rajput
Member Since 04 Feb 2013Offline Last Active Mar 13 2013 01:44 AM
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In Topic: PCR primer usage (Clonning & cDNA)
13 March 2013 - 01:45 AM
In Topic: In-situ hybridization
13 March 2013 - 01:34 AM
In situ hybridization is used for specific mRNA localization in tissue and a method of tissue specific expression for a gene.
Very useful for promotor studies as you have no need to tag your gene with gus or other sequence, then no further transformation in model plant and screening and then expression studies.
You can apply direct and save your 1-2 years time with In situ hybridization.
Good luck
Dr. Rupesh Kumar Singh
PhD (Plant Biotechnology)
Department of Biotechnology
Kumaun University, Nainital,
Uttarakhand, India-263139
Mob- +91-9911896375
Very useful for promotor studies as you have no need to tag your gene with gus or other sequence, then no further transformation in model plant and screening and then expression studies.
You can apply direct and save your 1-2 years time with In situ hybridization.
Good luck
Dr. Rupesh Kumar Singh
PhD (Plant Biotechnology)
Department of Biotechnology
Kumaun University, Nainital,
Uttarakhand, India-263139
Mob- +91-9911896375
In Topic: too much DNA on agarose gel
04 February 2013 - 04:32 AM
DNA run fast only when you increse current,
keep the voltage low and it will run slow,
very simple
it does not depend on quantity of DNA or sample,
it matter with size of DNA (bp) and respected voltage in aparatus
chears
keep the voltage low and it will run slow,
very simple
it does not depend on quantity of DNA or sample,
it matter with size of DNA (bp) and respected voltage in aparatus
chears
In Topic: Electrophoresis problem , please help pals !
04 February 2013 - 04:20 AM
Hello Friend
In this problem with gel electrophoresis, you need to check the gel apparatus and current flow in the assambly.
Second thing is your running buffer and pH of the buffer.
You need to prapare new TAE buffer with apropriate pH and then run your product,
Load small amount of Dye in a wel, without sample which will be a marker or control,
Hope your product will run in a proper way and resolve with marker.
chears
In this problem with gel electrophoresis, you need to check the gel apparatus and current flow in the assambly.
Second thing is your running buffer and pH of the buffer.
You need to prapare new TAE buffer with apropriate pH and then run your product,
Load small amount of Dye in a wel, without sample which will be a marker or control,
Hope your product will run in a proper way and resolve with marker.
chears
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