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Troubleshooting with cloning

Cloning

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#1 cspb

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Posted 04 March 2015 - 08:43 AM

Hi scientific community,

I am have a lot of troubles in cloning my vector in an insert. Their sizes are 6473bp and ~1kb, respectively. I am trying to have colonies with this ligation from several weeks. I am trying with 50ng and in a ratio of 1:7. When I got colonies the sequencing results were not as I expected.

 

Can someone help me optimizing transformation or ligation? 

 

Thank you.



#2 bob1

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Posted 04 March 2015 - 01:14 PM

More details needed: what sites are you using? How much are you ligating in what volume? how did you prepare the DNA insert? are you sure your digests are working? Are you sure the things you are working with are what they are supposed to be (e.g. you didn't mix up your backbone with another)?

 

What have you tried so far?



#3 christy

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Posted 04 March 2015 - 04:06 PM

Hello,

 

Try this link for ligation calculation http://www.insilico..../Lig_Input.html

 

what kind of ligation it is, sticky or blunt? Did you do self ligation. How long you are doing ligation and what temperature you are incubating. After ligation, did you screen your clones before sequencing.

 

 

All the best !!!







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