DNA Cloning Procedure

[begemot]

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Hey I need to prepare for an interview for tomorrow and i havent done this stuff in a long time, so could you please tell me briefly how you do this:
you take a sample of human tissue and you want to extract dna from it. How do you filter it from rna and other stuff?
Then you want to isolate a piece of dna. I assume you do that by using a pcr sequence to hybridize with denatured dna. Then once you've isolated I kind of remember that you would place that sequence into a bacterial plasmid for cloning.
But what if you don't know the sequence of that gene that you want to isolate, i.e. it is mutated or methylated as in cancer. How do you isolate it then? Thanks.

 

[298609]

well i usually start by doing a buccal swab to collect dna. then i make sure everything is spun down, etc, before moving on to a sequence of washes and treatments. i continue on to west philadelphia, where i was born and raised i do spend most of my days, however i do make sure to be chilling out, maxin all cool shootin some bball outside of the school. when a couple a guys who were up to no good, started making trouble in my neighborhood. i got in one little fight and my mom got scared she said you moving with your auntie and uncle in bel air. i whisteled for a cab and when it came near the license plate said...


and then you have a clone.

 

[bold text]

well i usually start by doing a buccal swab to collect dna. then i make sure everything is spun down, etc, before moving on to a sequence of washes and treatments. i continue on to west philadelphia, where i was born and raised i do spend most of my days, however i do make sure to be chilling out, maxin all cool shootin some bball outside of the school. when a couple a guys who were up to no good, started making trouble in my neighborhood. i got in one little fight and my mom got scared she said you moving with your auntie and uncle in bel air. i whisteled for a cab and when it came near the license plate said...


and then you have a clone.

NICE EXECUTION! 👍
For a minute there, I thought you were going to explain PCR.

 

[bold text]

...How do you filter it from rna and other stuff?...

RNAse, Protease ect. then centrifugation.

But what if you don't know the sequence of that gene that you want to isolate, i.e. it is mutated or methylated as in cancer. How do you isolate it then?

Take the DNA, cut it with a nuclease a little bit at a time until you have the gene of interest intact on the smallest piece of DNA possible (after each cut, test to see if the gene is expressed). Sequence.

 

[begemot]

RNAse, Protease ect. then centrifugation.



Take the DNA, cut it with a nuclease a little bit at a time until you have the gene of interest intact on the smallest piece of DNA possible (after each cut, test to see if the gene is expressed). Sequence.

So if i cut dna at some point around a gene of interest, i then need to transform that onto a plasmid to see if it will produce a functional protein? And then I sequence that or do some other test, i.e. to see if it's methylated at some nucleatide? Thanks!

 

[bold text]

So if i cut dna at some point around a gene of interest, i then need to transform that onto a plasmid to see if it will produce a functional protein? And then I sequence that or do some other test, i.e. to see if it's methylated at some nucleatide? Thanks!

exactly.

 

[Amrazzz]

Uh, will making cDNA help?

Homogenize tissue with TriReagent. Allow for it to sit, centrifuge @ 4deg C for 3 minutes. Extract clear top layer (this is the RNA) into another tube. Add isopropyl alcohol, centrifuge @ 4deg C for 15 minutes. Remove supertanant, add EtOH, vortex slightly. Centrifuge @ 4deg C for another 10 minutes. Remove EtOH, allow for it to dry.

Quantify RNA (if you want specific amount), otherwise add proportional amount of genomic DNA wipeout buffer followed by reverse transcriptase, RT buffer, and Primer mix. Heat to approx 53 deg for 15 minutes, and then to 92 for 3 minutes. Voila cDNA....

Follow up with PCR for gene of interest.

 

[LebLlama]

you already have a med school interview?

 

[Geekchick921]

well i usually start by doing a buccal swab to collect dna. then i make sure everything is spun down, etc, before moving on to a sequence of washes and treatments. i continue on to west philadelphia, where i was born and raised i do spend most of my days, however i do make sure to be chilling out, maxin all cool shootin some bball outside of the school. when a couple a guys who were up to no good, started making trouble in my neighborhood. i got in one little fight and my mom got scared she said you moving with your auntie and uncle in bel air. i whisteled for a cab and when it came near the license plate said...


and then you have a clone.

I think you may be hearing from bleargh's lawyer. 😉

you already have a med school interview?

It's probably for a research job, maybe for the summer.

 

[MisterVeil]

But what if you don't know the sequence of that gene that you want to isolate, i.e. it is mutated or methylated as in cancer. How do you isolate it then? Thanks.

If this is the case, you probably don't know a portion of the sequence (ie, the mutated part). You can still design primers for the gene (ie, primers that correspond to various parts of the gene's sequence) and then run a sequencing reaction (dideoxy sequencing method).