isolation of genomic DNA - (Jul/06/2008 )
lets take it step by step.
AIM : We are to PCR carbonic anhydrase gene (300bp) from the cheek cells.
1 Collect cheek cells with cytology brush.
2 Put brush in 0.5ml TEMPLATING BUFFER + vortex (all in 2ml appendorf)
3 Centifuge n discard sup.
4 Add 300ul TEMPLATING BUFFER n resuspend pellet
5 Add 0.02 ml PROTEASE to digest cellular material.
6 Put appendorf 55 digre celcius for 1 hr for protease to work.
7 After 1 hr put appendorf at 95 dig cel for 20 min to inactivate protease.
8 Centrifuge to pellet any debris. therefore i understand sup has the DNA.
9 collect the DNA n put in -20 for further use.
now go for the PCR stuff i.e. making the master mix and all that.
My questions are:
1) if we are isolating the whole genomic DNA, then how can we see the 300bp carb-anhydrase gene in the 2% gel. we have not digested the genomic DNA in any ways. can any1 see any anomalies in this protocol that i have given here..
2) TEMPLATING buffer is made of SDS and NaCl and EDTA correct??
3) is the general procedure same for extracting genomic DNA from any type of tissue. i know that the ingredients differ, but is the funda the same..?
RESULT was positive.. dont ask me how..
AIM : We are to PCR carbonic anhydrase gene (300bp) from the cheek cells.
1 Collect cheek cells with cytology brush.
2 Put brush in 0.5ml TEMPLATING BUFFER + vortex (all in 2ml appendorf)
3 Centifuge n discard sup.
4 Add 300ul TEMPLATING BUFFER n resuspend pellet
5 Add 0.02 ml PROTEASE to digest cellular material.
6 Put appendorf 55 digre celcius for 1 hr for protease to work.
7 After 1 hr put appendorf at 95 dig cel for 20 min to inactivate protease.
8 Centrifuge to pellet any debris. therefore i understand sup has the DNA.
9 collect the DNA n put in -20 for further use.
now go for the PCR stuff i.e. making the master mix and all that.
My questions are:
1) if we are isolating the whole genomic DNA, then how can we see the 300bp carb-anhydrase gene in the 2% gel. we have not digested the genomic DNA in any ways. can any1 see any anomalies in this protocol that i have given here..
You will see the 300bp product after doing PCR on this genomic DNA.
2) TEMPLATING buffer is made of SDS and NaCl and EDTA correct??
Your templating buffer is your own unique buffer, check the recipe. However, the components look right for this PCR purpose.
3) is the general procedure same for extracting genomic DNA from any type of tissue. i know that the ingredients differ, but is the funda the same..?
Procedure is not the same, but this seems like a good protocol for doing PCR directly from lysate, rather than going to all the trouble of isolating pure genomic DNA, when your purpose is merely to do a small, simple PCR.
RESULT was positive.. dont ask me how..
You should ask yourself how. There is a logic behind teh success of each step and experiment and you need to know. Talk with a senior colleague and ask questions about importance of each step and each reagent. And READ stuff.