I have a purified plasmid having the insert of 655 bp. when i am pcr amplifying it with gene specific primer ideally i should get single amplified band of size 655 bp but after running it on gel i got two bands one is of almost size of vector back bone and other is of 655bp. what is wrong in this? Thanks in advance for useful suggestions.
Difficult to say without the pcr conditions and length of the plasmid but I can imagine with a short plasmid and long annealing and extension times then a second larger and weaker band might be generated by each primer extending right round the plasmid and insert and producing an ampliner of size insert plus backbone.Is this possible. Can we see a gel image please?. How much template dna did you amplify...it might be a visible amount again weaker than the short amplimer. What cloning method did you use. If it were possible to clone 2 inserts into the vector in opposite orientations again a full length amplimer might be possible
All the temperatures of annealing , denaturation and extension should be made correctly, if possible changed and then see the results. If you send the pic of the gel it would be much clear to identify.
Hello Smita
The only reason is you are using too much template therefore you will see the template in addition to your PCR product on the gel. If you are using a plasmid as a template you should dilute it (around 100-200 times) then use it as a template. It was my experience too.
Good luck
Thank you all for your answers. I am following the standard cloning protocol with double restriction digestion with EcoRI and Xho I then gel extraction of both the vector and insert then ligation with T4 DNA ligase with 1:3 vector insert ratio. Also before proceeding to cloning i have checked my primer efficiency and all temperatures to get best amplification through gradient PCR. May be I am using too much concentrated plasmid as the template as said by Ali Javadmanesh . Template plasmid is of 394 ng/ul and i have used 1 ul for pcr reaction.
Below I have attached the gel pic. Lane 4 in the gel pic is the amplified product after PCR.
You can expect to see 10ng or more dna on an agarose gel so if your template concentration is 394ng/ul the large band is certainly original template.Running an equivalent dilution of non amplified plasmid template should give you the same intensity large band without the small pcr product as confirmation
Hi,
It might be the buffer or the pcr set are contaminated with other DNA. Try to use new buffer and new pcr set.
Thank you all for your suggestion. I came to know what my problem was. The problem was with one of the restriction enzymes which i was using for double digestion hence the insert did not fall off.
- Badges
- Science method