First of all, you need to have the gene sequence you want to study, you can get it from the pubmed (I can help you with that if you want). Then you should design a forward and a reverse primers to amplify taht gene by PCR and finally, you will sequence the amplification. Is not hard at all!

Which is your biological sample? Is it blood? You can get a DNA extraction kit from blood. But, I repeat, first of all you need to know your sequence of interest and start from there. Good luck!

Thanks Ms Barbara Granata

the sample is blood

i am looking at TG gene, TPO gene and ZFAT1 gene

Where can i get the sequence?

Where can i get the detailed protocol?

As Barbara say, you can get the sequence at NIH or for example also at ENSEMBL http://www.ensembl.org/Homo_sapiens/Info/Index

For a protocol you have to search only for PCR standard protocol.

Hi,

As above mentioned details,

1. Gene information. the nucleotide sequence for using as a reference seq when you do sequencing analysis plus the basic information for designing primers.

You need to download from NCBI or ENSEMBL.

For ex. TG for homosapiens, you need to see which isoform, if you dont know which isoform to take, then take the longest one covering all the exons.

http://www.ncbi.nlm.nih.gov/gene/7038 (for your TG gene)

2. Mark exons in the sequence, design primers may be using Primer3 online designing tool. Order them

3. PCR, basic protocol for PCR and primer desgn you will get in most of the places.

4. You need to sequence the PCR amplified products of each exons of genes ypu mentioned. Compare with the ref. sequences you downloaded and look for variations in them. Check databases in NCBI like dbSNP or 1000 genome project in ENSEMBL..etc.

Good luck

hey,

For extraction you can use qiagen DNA extraction kit (there are several other kits available in the market). after extraction, you need to do PCR amplification.you can use the simple program of Initial Denaturation: 95oC/ 3 min 35 Cycles: 95 oC / 30 min, 59 oC / 1 min, 72 oC/ 1 min, Final Extension 72 oC - 5 min. you can change the PCR program as per your need. After PCR you may cleanup the PCR product by Na-Acetate precipitation method. then you have to do sequencing PCR. Again you have to clean up the product by Na-Acetate precipitation method. then heat chilled your product with 10 ul of hidi. then load it in the sequencer.

btw, TG Gene ID: 7038, TPOGene ID: 7173, ZFAT Gene ID: 57623.