4 replies to this topic

[siddharthsameer]

Hello everyone
I am in great problem and i need the help of you people.
I am trying to clone but it failed.
I am using insert of 1.4kb of psedomonas aergonisa and using pPICZalphaA as a vector which is 3.6kb, I got the correct pcr band, and when I double digested my vector and insert  i had the correst band on the restriction dgestion, performed ligation , and tranformed into E.coli competent cell. got colonies , when i did the midi prep, and alkaline lysis method, and when determined the concentration of DNA i had good DNA concentration but when I tried to run on agarose no bands were visible.
As my tenure of thesis has finished and I am asked to stop at this point..
I dont know the reason why it did not work, it would be really nice of some people who could help me in this...
I am attaching the complete information of my experiment
Kindly reply and suggest me what reason can i give for the failure of my project
thanks and regards

Attached Files

•  cloning information.docx   113.65K   173 downloads

[bob1]

It's possible that you degraded the DNA during the isolation - it is critical that the alkaline lysis step is as short as possible (definitely less than 5 min) to prevent damage to the DNA.  Digested DNA still gives signal on a spectrophotometer, but won't give you bands.

How much DNA did you run on the gel - staining method??

Did you check for the presence of an insert?

[siddharthsameer]

thank you so much for your reply I used 10microlitre to run on agarose but it did not show, i tried midi and mini and maxi prep using the kit also, and along with I tried other sample from my lab and i worked fine and showed the band.. Since after the transformation I had plenty of colonies  and the I chose 7-8 colonies from the plate and did plasmid prep and then agarose , but no band so I cant send it for sequencing ...
since my period is over for the practical they asked me to write why did it fail to clone keratinase sequence, what could be the reasons and what could be done in the future to avoid IT...kINDLY GIVE ME SOME IDEA  BECAUSE I HAVE NO EXPERIENCE IT CLONING S I HAVE TO WRITE ...i WOULD BE THANKFUL AND GREATFUL TO YOU, WAITING FOR YOUR REPLY..

[HOYAJM]

bob1, on 25 October 2012 - 12:32 PM, said:

It's possible that you degraded the DNA during the isolation - it is critical that the alkaline lysis step is as short as possible (definitely less than 5 min) to prevent damage to the DNA.  Digested DNA still gives signal on a spectrophotometer, but won't give you bands.

How much DNA did you run on the gel - staining method??

Did you check for the presence of an insert?

Bob has given you an excellent possibility of where you went wrong. Colonies should have yielded LOTS of plasmid DNA, but you got no DNA. Thus, your plasmid isolation was faulty.

You can explain there are other methods for screening colonies like colony PCR for an insert-specific product.

[siddharthsameer]

HOYAJM, on 26 October 2012 - 06:38 AM, said:

bob1, on 25 October 2012 - 12:32 PM, said:

It's possible that you degraded the DNA during the isolation - it is critical that the alkaline lysis step is as short as possible (definitely less than 5 min) to prevent damage to the DNA.  Digested DNA still gives signal on a spectrophotometer, but won't give you bands.

How much DNA did you run on the gel - staining method??

Did you check for the presence of an insert?

Bob has given you an excellent possibility of where you went wrong. Colonies should have yielded LOTS of plasmid DNA, but you got no DNA. Thus, your plasmid isolation was faulty.

You can explain there are other methods for screening colonies like colony PCR for an insert-specific product.

thanks but i tried all methods of plasmid isolation,but the results were the same, can you give some idea how can i write about all this.. thanks and regards