I am try to delete a domain containing 660bp using Takara PrimeSTAR Mutagenesis Basal Kit. However, sequencing results show no success in deletion of this region. Is the size too big for deletion through this method?
Dear KeeSIang
In theory there is no limit to the delection size that you can perform it those regard a gene cloned into a plasmid. Of course is important that deletions do not involve regions of the plasmid essential for vector replication, mantainance (e,g replication origin, antbiotic resistance)
Do you have colonies but the colonies corresponding from the DNA template?
How much dna template did you used for the PCR?
How many colonies did you sent to sequencing?
Generally it is possible if you use to much DNA that this is able to give you colonies of background.
Generally for mutagenesis, deletion of insertion i'm not use a kit but i'm using the PIPE cloning (Method PIPE (Polimerase incomplete primer extention) method for enz...
)and to minimize this problem i'm using 0,1ng of template (using Kapa hifi polimerase that is very processive and able to provide a strong amplification also with so low amount) and the PCR is followed by 1h digestion with dpni to digest it.
good luck
Manuele Martinelli
Do you have colonies but the colonies corresponding from the DNA template?
--> I do manage to get colonies but not many, less than 20.
How much dna template did you used for the PCR?
--> 100pg
How many colonies did you sent to sequencing?
--> 6 so far
Perhaps the template is too much for the PCR as my sequencing data show the colonies express the native sequence.
I will study the figure. Thank you very much for your advice.
DEar KeeSiang
some more question:
How big is your plasmid? Did you check in agarose gel the PCR product before transformation?
there is no limit, but more than 100bp is little standardization need. i have done around 300 bp using algilent site-directed mutagenesis kit for my experiment.
if you used 60 degree in yr experiment try like 64 degree
increase your time in pcr cycle depending on your plasmid length
most of the time u wont see in agarose after dpn digestion. So transform isolate and run agarose with your control (start plasmid)
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