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Help cloning large insert into large vector


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#1 itsmyapple

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Posted 19 July 2010 - 07:21 AM

Hi,

I am currently trying to clone a large insert of 7.5kb into a large vector of 12kb. My current protocol is to excise the insert from pBluescript using pac1 and asc1, gel extract using qiagen kit, phenolchloroform extract and precipitate and then ligate into the vector which has also been digested with pac1/asc1, treated with SAP and phenol chloroform/ chloroform extracted and precipitated. The ligation is done using the roche rapid DNA ligation kit at 20 degrees for 5 minutes and a 3:1 insert:vector ratio. I then transform into dh5a cells from invitrogen.
I have tried this multiple times and get very few colonies around 12 per plate. I have grown up these colonies, mini preped and digested them and find that i get a multitude of strange bands on the gel which dont correspond to empty vector but done correspond to my finished vector either.
I have used this method to clone a smaller fragment of 3kb into the same vector which is worked so i think the problem is with the size of the insert. I have heard that xl-10 gold cells are better for large vectors so i am considering trying them instead? I am also considering using a different ratio of insert:vector in my ligation reaction? If anyone can help me and tell me if these are good ideas or offer any insight into my problems I would really appreciate it.

Thanks

#2 perneseblue

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    Unlimited ligation works!

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Posted 19 July 2010 - 06:10 PM

Let me guess, the strange inserts you recovering are also smaller than your 7.5kb insert? Even if the ligation is poor, a proper ligation should yield 100s of colonies.

I would suggest that you stop using rapid DNA ligation kit. Those thing are bad news for ligation of large fragments.

I would suggest that you use normal T4 DNa ligase buffer and ligate your vector+ insert overnight at 4 C.

Is the backbone of your vector a high copy plasmid? In my opinion the final size of your vector (20kb) is very near the edge of what a high copy plasmid can safely carry. A BAC backbone would be more suitable for plamids this big.

When you gel extract your vector and insert, are the DNA fragment exposed to UV light for a long time (more than 1 min). UV light is damaging to DNA fragments, so best to minimize UV exposure time. Adding 1mM (final concentration) of guanosine into the agarose gel will help protect your DNA from UV damage.

What method of transformation are you using? Chemical or electroporation? Electroporation is far superior to chemical transformation when introducing large plasmids.

And yes, e coli strains do make a difference. DH10 alpha`is good. I have not used Xl10 before. When you start growing up the colonies in culture, use a lower growing temperature eg 30C. A rich medium like SOC also helps.
May your PCR products be long, your protocols short and your boss on holiday

#3 itsmyapple

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Posted 26 July 2010 - 04:41 AM

Hi,

Thanks for getting back to me.

I have tried a couple of your suggestions such as using t4 ligase overnight rather than the rapid ligation kit. I am now getting far more colonies 100-200 per plate. Many of these colonies are showing up positive in a colony PCR using m13f sequence present in vector and a reverse primer against a site 1.2kb into my insert. I have been growing up cultures at 30 degrees and mini-prepping and digesting. The results of the digest show inserts of about 3.5kb, not 7.5kb as expected.

Can anyone give me further help on what is wrong?

thanks

Edited by itsmyapple, 26 July 2010 - 04:42 AM.


#4 perneseblue

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Posted 26 July 2010 - 04:22 PM

I will try my best.

1- Did you gel purify both the insert and vector?
If you did not, please do so. Small plasmid are far far easier to transform compared to large plasmids. So the colonies that you recover tend to contain small plasmid... containing for instance truncated PCR products, or random bits of contaminating bacterial genomic DNA which are ligatable because they have been cleaved the RE used to prepare either the insert or vector

2- If you did gel purify your insert... it gets more difficult.
-run some of your cut insert and vector on a ge (use the narrowest well you can make)l, do you see any extra bands that is not suppose to be there? If there are contaminating bands, gel purify the vector and/or insert a second time

If all is clean... could you tell me more about this insert? How was this insert made (PCR or cut oout from another plasmid) and what does this insert contain. More specifically does this insert contain any genes that are expressed in prokaryotes.
May your PCR products be long, your protocols short and your boss on holiday

#5 itsmyapple

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Posted 27 July 2010 - 07:21 AM

The insert has been gel extracted but the vector hasn't it was just digested, treated with sap, phenol/phenol chloroform extracted and precipitated. Both show up as single bands when ran on a gel.

Each end of the insert was orinally generated by PCR, these were then cloned into pBluescript before using recombineering to generate the full insert.

thanks

#6 perneseblue

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Posted 27 July 2010 - 03:38 PM

The insert has been gel extracted but the vector hasn't it was just digested, treated with sap, phenol/phenol chloroform extracted and precipitated. Both show up as single bands when ran on a gel.

Each end of the insert was orinally generated by PCR, these were then cloned into pBluescript before using recombineering to generate the full insert.

thanks


And all the bands (insert and vector) are of the right size?

It is possible that the contaminant is coming from the vector. gel purify the vector. Try to keep the UV exposure to a minimal. It has been reported that 1mM of guanosine help protect DNA from UV damage.

Lets see if gel purification of the vector will help.

Still.. you are getting a positive signal from colony PCR.... Can I have a look at that gel. If the PCR is real, it means that you are getting insertion... from truncated insert fragments that are coming from somewhere.

Does your insert have any gene expressed in bacteria? Big difficult repeat structures?
May your PCR products be long, your protocols short and your boss on holiday

#7 laborat

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Posted 12 January 2011 - 10:12 PM

could you please tell me if you could ligate your 12kb plasmid with your 7 kb insert and transform in dh5a. The reason I am asking you this is, I too am using same backbone vector and have been experiencing similar failures. just wanted to know if you succeeded. if you did succeed i would be happy if you can share some tips about this.



Hi,

I am currently trying to clone a large insert of 7.5kb into a large vector of 12kb. My current protocol is to excise the insert from pBluescript using pac1 and asc1, gel extract using qiagen kit, phenolchloroform extract and precipitate and then ligate into the vector which has also been digested with pac1/asc1, treated with SAP and phenol chloroform/ chloroform extracted and precipitated. The ligation is done using the roche rapid DNA ligation kit at 20 degrees for 5 minutes and a 3:1 insert:vector ratio. I then transform into dh5a cells from invitrogen.
I have tried this multiple times and get very few colonies around 12 per plate. I have grown up these colonies, mini preped and digested them and find that i get a multitude of strange bands on the gel which dont correspond to empty vector but done correspond to my finished vector either.
I have used this method to clone a smaller fragment of 3kb into the same vector which is worked so i think the problem is with the size of the insert. I have heard that xl-10 gold cells are better for large vectors so i am considering trying them instead? I am also considering using a different ratio of insert:vector in my ligation reaction? If anyone can help me and tell me if these are good ideas or offer any insight into my problems I would really appreciate it.

Thanks



#8 itsmyapple

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Posted 13 January 2011 - 02:43 AM

Hi,

I did not succeed in cloning my insert into my vector but someone in my lab did manage it. As far as I know they didnt do anything particularly special I think it was just perserverence and a bit of luck. Sorry I couldn't be more helpful. Goodluck.




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