PCR and Gene cloning

[FFK]

Hello I'm just starting to learn molecular biology. I hope someone out there able to explain the difference between gene cloning with plasmid and Polymerase chain reaction (PCR). Are they have the similar function (to amplify the DNA fragments) but work at different way? Thank you.


Regards,
Foo

[aftabac]

Hi
yes, both ways are used to amplify desired gene or DNA Fragement. but there is difference in application as by PCR DNA is amplified to analyze it but by plasmid cloning, DNA is stored for long time, analyzed and also used for expression in experiments. these are main difference, although there are planty of other differences.

regards
aftab

[FFK]

Thank you for sharing =). Neway, I've one more question here. Can we say microsatellite is the primer uses in PCR complementary with the known sequences? Thank you


Regards,
Foo

[hanami91]

PCR targets a segment of DNA with specially designed primers to ampifly it through a series of cyles in the thermocycler. The primers are typically @ 20 bases long and target the 5' end of each strand. In the first stage of a PCR cycle the strands are denatured, -heated (@94C) to disrupt the hydrogen bonds and they separate. In the second stage-annealing the temp lowers (@ 60-70C) to just below the primer melting point and the primers fix to their position on the DNA template. During elongation (@72C) the strands are elongated by the base supplied with the PCR reaction.

In gene cloning with plasmids, bacteria are transformed. Usually E. coli, to take up a desired plasmid which carries a desired genetic trait, and another trait used for selection such as a specific antibiotic resistance. The transformed bacteria are cultivated for a few days and then spread on petri dishes containing the antibiotic the plasmids are resistant to. Bacteria that grow on this have the plasmid. Colonies are then selected for pure cultures. From the pure cultures the plasmids can be extracted from the bacteria through miniprep.
Hope this was helpful,
haanmi91

[admin]

Thanks Hanami

[thebetafemale]

I understand these answers. They describe how the procedures are done. But it still does not really explain what the difference in purpose is.

PCR is when you target a specific area along a chromosome using primers that attach to single stranded DNA on each end of the desired area. That area is copied.

In cloning you put a DNA fragment into a plasmid through a ligation process, then put the plasmid into chemically competent bacteria. When the bacteria divide and reproduce, the plasmid gets copied and therefore, so does the DNA fragment you inserted into the plasmid.

From the way I see it, the difference is that PCR can be used on whole genomic DNA to obtain a fragment in the first place, while cloning requires that the DNA fragment is already isolated from the genomic DNA.

I'm new at this, too, so please feel free to correct me.

[TuanVo]

Hello all,
I study about cloning. My gene are too long (about 5,200 bp). I use Phusion DNA polymerase to amplify this gene but I got many wrong bands and right band was very thin. I would like to reduce wrong bands and increase my target band. Please help me.

Thank you very much
Best regard

TuanVo

[mmorgan]

Originally Posted by TuanVo

Hello all,
I study about cloning. My gene are too long (about 5,200 bp). I use Phusion DNA polymerase to amplify this gene but I got many wrong bands and right band was very thin. I would like to reduce wrong bands and increase my target band. Please help me.

Thank you very much
Best regard

TuanVo

Hi,
Try purifying the 5.2kb band and using that as template for PCR with the same primers (i.e. re-amplify it).

If your initial template is cDNA this usually helps because it greatly reduced the complexity of your template sample. However, if you're amplifying from plasmid I don't think this will make a big difference, in that case start playing with the PCR conditions.

Another way to do it is to split the gene into two halves, PCR the halves independently and then stitch them back together again.

[pc88]

Hi everyone! I have read the MIQE guidelines for qPCR and the article refers the importance to assess the annealing of primers with suitable RNA regions (e.g: without hairpins). My question is if there is a good software to overlap primer sequences with RNA secondary structure? Moreover, I though that secondary structures were denaturated with the high temperature cycles... if that's the case... it doesn´t make sense my previous question xD

Can anybody help?

thanks