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PCRed-vector approach to tag switching


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#1 Trof

Trof

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Posted 11 February 2019 - 06:53 AM

Hi,

we have a very limited lab in the means of molecular cloning in general, and some access to a lab of a good-old-cloner guy and I'm looking for a quickest way how to switch our plasmids from His to FLAG (DDK) tag.

I had no say in the original order, so 2 our genes are now in simple pcDNA3(+) vector with C-His, and it doesn't work well in WB, His antibodies suck.

 

Original idea was to cut out the 6His part and order oligos to make a cassette with FLAG to insert instead. But as the gene was put into MCS with lots of cut sites around, the His tag has NO sites on 3' end whatsoever (at least not in less than 1kb distance with an enzyme cutting less than 4 times..). Seems like good-old cloning days are gone and people do not create vectors with RE.

 

The other option was to PCR the whole vector (minus His), and add primer overhangs to any enzyme site I wish to have there (plus some additional bases to ensure cutting) and use a proofread polymerase to amplify it, then cut the bits off and insert the cassete.

The "good-old-cloning" guy I'm m consulting this with said Phusion is slow for this (plasmid is around 8kb long), but polymerases improve every year, so I though that some Phusion or something could do.

Also, I do remeber someone here swearing on PCRing vectors just as a common way to prepare them, ready to be cut and ligated. If you are the guy (I'm sorry to forgot who it was), what do you use?

I have no prior experience with this and in case of Phusion and other more "luxury" stuff I need to buy it first (which takes some time). But it is still cheaper than just order the genes anew from Origene with DDK, probably, and maybe even quicker too.

Any suggestions? Experiences?

Thanks.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#2 Trof

Trof

    Brain on a stick

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Posted 11 February 2019 - 07:59 AM

Huh, I just totally ignored the option to just use the RE site just before the His tag and insert a casette there with stop codon, leaving the old His there untranscribed. Well, I'll still keep the PCR approach as a option :)


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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