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siddharthsameer

Member Since 12 Jul 2012
Offline Last Active Nov 05 2012 12:49 AM

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Active Posts 36
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Location
braunschweig, germany

About me

My research interests
molecular biotechnology especially molecular cloning

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06 Sep 2012 - 16:01
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Topics I've Started

cloning into pPICZalphaA fails

25 October 2012 - 01:51 AM

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

molecular cloning failure

23 October 2012 - 05:18 AM

Hello
I am a master student and I am doing my master thesis in the cloning of keratinase sequence in competent cell.I tried several different methods and procedure for the cloning and I got the colonies but when I did the mini prep I had the good concentration of the DNA but when run on agarosei could not see the band.. I have to stop my thesis at this point but I am unable to think What could went wrong for the failure of the cloning, I am really confused what reasons should I put it in my discussion and in the overview that what should be done in future to avoid this proble.. Any help and answer would be of great help to me... thanks and regards

Ligation

07 September 2012 - 02:38 AM

Hello everyone
I need suggestion and advice for the ligation
I want to ligate 1,4 kb insert with the vector pPICZaplha A(3,6kb) and transform into E.coli competent TOP10: agter the gel extraction the concentration of my PCR product (insert) is 9,15ng/µl and for the vector is 30,5ng/µl. Are my concentration fine to carry out the ligation?
I am here to take the advice because last time I had more or less the same concentration my both insert and vector but after the transformation I didnt get any colobnies..So can anyone suggest me the correct manner to carry out the proper ligation.. i am stuck up with the 4 months.. Kindly suggest me how should I carry it out to get the result.
Last time i had the molar ration of 2:1,3:1,4:1 but noone of them worked..
Waiting for the quick reply thanks and regards

QIAquick Gel Extraction

06 September 2012 - 03:49 AM

hello everyone
I have a small doubt regarding the gel extraction purification. I cut of my fragment from the agarose and after weighing the weight of my fragments are 460mg and 550mg..but the Extraction kit says for the slice of more than 400mg we should use more than one QIA column.I have never used in such conditions.You can anyone suggest me how to proceed with my extraction. I am unable to figure it out..
Quick reply would be really appreciated..
regards

agarose gel electrophoresis

06 September 2012 - 12:18 AM

I forgot to add ethidium bromide in the molten agrose however i added the ethidium bromide to the running buffer...Will i be able to visualise the band, if not how caqn I rescue it. kindly let me know..
with regards

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