13 replies to this topic

[Teagan27]

Hi,

I was wondering if anyone else out there has used the In-fusion PCR Cloning kit from Clonetech?

I have been attempting (since April) to clone a small gene (600bp) into pFLAG-CMV-2 (N-Terminal Flag tag) and pFLAG-CMV-5.1 plasmids (c-terminal FLAG tag) (sigma)... but is has proved to be very difficult with this kit.

Firstly we designed the primers for a single restriction site. This meant that the vectors simply re-joined (cut with Not1 and Sma1). We did find one clone in the vector cut with Sma1..after much searching (screening 100's of colonies).. only to discover after sequencing that it had several mutations as we had used the wrong polymerase (oh the silly things we do).

We then redesigned the primers to clone into the vector after it had been double digested, as this is meant to be more efficient as the vector will not simply re-join (oh and got a cloning grade polymerase ). We digested the pFLAG-CMV-2 vector with Not1 and BAMH1 and the pFLAG-CMV-5.1 vector with Sma 1 and EcoR1.

This again failed as the double digests were not efficient and the vectors which were only digested once simply re-joined and after screening of 100's of colonies we did not find any clones what so ever.

We did the digests sequentially and also cleaned up the plasmid DNA in between digests. We also made sure the restriction sites were as far away as possible and there was sufficient enzyme and buffer. Even after these factors were considered we still had a high back-ground of vector which was not double digested (which we checked by doing a ligase reaction).

We have purchased a pre-linearized pFLAG-CMV-2 vector from sigma, which should solve our problem for one of the constructs... but while we are waiting for it to arrive we thought we would attempt to linearized both the vectors by PCR.. but again this is failing.

I do understand that this kit is good, but a low back ground of undigested is extremely important. If anyone else has had some experience with this kit then i would like to hear about your successes or failures.. as it may help me in my epic quest to construct these vectors.

help me!

Cheers Teagan

[tea-test]

hi, i used this kit already for cloning three inserts into a pCMV vector. all of them worked and I got about 1 positive clone per 20 colonies just following the guidelines and without any optimisation.

it is very important that your primer design is correct. there is a tool for this on the clontech website but i think you used it anyway.

another danger is (undigested) template vector in your PCR reaction. either gel purify your insert or clontech recommends to treat the PCR reaction with DpnI.

IMHO another possibility to overcome this problem would be to use template and target vectors with different antibiotic resistance genes.

Concerning the undigested target vector i think it is better to do a single cut and gel purify the linearized vector. you won't be able to separate single and double cut vectors if the RE sites are relatively close together.

I hope this was helpful.

[Teagan27]

tea-test, on Aug 14 2009, 08:55 PM, said:

hi, i used this kit already for cloning three inserts into a pCMV vector. all of them worked and I got about 1 positive clone per 20 colonies just following the guidelines and without any optimisation.

it is very important that your primer design is correct. there is a tool for this on the clontech website but i think you used it anyway.

another danger is (undigested) template vector in your PCR reaction. either gel purify your insert or clontech recommends to treat the PCR reaction with DpnI.

IMHO another possibility to overcome this problem would be to use template and target vectors with different antibiotic resistance genes.

Concerning the undigested target vector i think it is better to do a single cut and gel purify the linearized vector. you won't be able to separate single and double cut vectors if the RE sites are relatively close together.

I hope this was helpful.

Hi,

Thank you so much for your reply.

So you only used a single RE site? This what i did orginally. One vector with Sma 1 (blunt ends) and the other Not1 and only got one positive colony with the Sma1 digested vector (only screened 50 out of 100's). We have been gel purifying the insert and the positive colony we got  had the insert in the right orientation.. but had 5 point mutations because of the super bad polymerase we used.. so i know everything is in order.. its just the efficiency of my double digest which is proving a challenge.

So this is why i have brought the pre-double-digested target vector and i am attempting to linearize by PCR... doing another infusion reaction on monday.. so hopefully this answers my prayers. If not i think i will go back to the single digest atleast for the Sma1 digested vector and just search and search because i know there will a clone somewhere! The Not1 digested vector will not be as sucessful.. i think because it has sticky ends its more favourable to religate as opposed to the blunt Sma1 (i cant use this RE in the other since it is far away from the FLAG Tag). Should know by the end of next week.. very frustrating.. because i have achieved nothing so far for my honours year. I think a ligase cloning system may have worked quicker.. better the devil you know.. than the devil you dont

Cheers, Teagan

[Teagan27]

Oh sorry, another question.. did you use the cloning enhancer? we have not been using this.

cheers

[swanny]

I, too, have had limited success with InFusion, both the Dry-down version and the Advantage. Although the chemistry seems pretty robust, there are a couple of critical steps. I really need the system to work well, as I'm trying to do 96 simultaneous reactions!

First of all, the vector digestion. As others said, gel purify to get rid of undigested, or partially digested vector (in the case of a 2-cut system). I had months of digestion pain, and I fixed it by growing the bacteria in LB rather than 2TY or Terrific broth (as had been suggested by a colleague). The cell pellet and plasmid yield will be lower in LB than the other rich media, but the digestion goes to very high percentage of completion every time. Not sure at the moment why this is so (presumably something - polysaccharide perhaps? - sticking to the DNA and inhibiting the digestion?), but I know it works, and at the moment, that's enough for me.

When I do InFusion-type cloning, I always transform my cells with digested vector before the actual DNA polymerase treatment stage. If I get significant numbers of colonies, I just redigest until the background is low, then I proceed to the fun bits.

Next, you have to get rid of dNTPs from the insert, so either gel purify or use the cloning enhancer. Enhancer seems to have DpnI, to digest template, but it must have something else that sops up the remaining dNTPs from the PCR. Remember the polymerases used in InFusion and LIC/SLIC only chew back the DNA 3' to 5' in the absence of dNTP: in early protocols you stop the reaction by adding one nucleotide.

If you keep on having no joy, think about a non-commercial system. Read these papers and see what you think: the supplementary data of the Bieniossek paper is just packed with good ideas and it is the best damned protocol paper I think I have ever seen! The document is so big, I can't attach it here!

 Li_and_Elledge_SLIC_2007.pdf   281.35K   233 downloads
 bieniossek_etal_2009.pdf   438.26K   487 downloads

All the best!

[Teagan27]

swanny, on Aug 17 2009, 09:50 AM, said:

I, too, have had limited success with InFusion, both the Dry-down version and the Advantage. Although the chemistry seems pretty robust, there are a couple of critical steps. I really need the system to work well, as I'm trying to do 96 simultaneous reactions!

First of all, the vector digestion. As others said, gel purify to get rid of undigested, or partially digested vector (in the case of a 2-cut system). I had months of digestion pain, and I fixed it by growing the bacteria in LB rather than 2TY or Terrific broth (as had been suggested by a colleague). The cell pellet and plasmid yield will be lower in LB than the other rich media, but the digestion goes to very high percentage of completion every time. Not sure at the moment why this is so (presumably something - polysaccharide perhaps? - sticking to the DNA and inhibiting the digestion?), but I know it works, and at the moment, that's enough for me.

When I do InFusion-type cloning, I always transform my cells with digested vector before the actual DNA polymerase treatment stage. If I get significant numbers of colonies, I just redigest until the background is low, then I proceed to the fun bits.

Next, you have to get rid of dNTPs from the insert, so either gel purify or use the cloning enhancer. Enhancer seems to have DpnI, to digest template, but it must have something else that sops up the remaining dNTPs from the PCR. Remember the polymerases used in InFusion and LIC/SLIC only chew back the DNA 3' to 5' in the absence of dNTP: in early protocols you stop the reaction by adding one nucleotide.

If you keep on having no joy, think about a non-commercial system. Read these papers and see what you think: the supplementary data of the Bieniossek paper is just packed with good ideas and it is the best damned protocol paper I think I have ever seen! The document is so big, I can't attach it here!

Li_and_E...LIC_2007.pdf
bienioss...tal_2009.pdf

All the best!

Hi,

Thank-you for your reply and for the papers. It is interesting what you have said about the digest. I have a feeling i used SOC media for my plasmid prep, which is high in glucose, so this may have been inhibiting my digestion. Did you do double digests? and did you do them seqentially?

Also when you have had success how many clones did you get per reaction? 100% or lower?

I have managed to linearize my vectors by PCR.. doing some infusion reactions today and hopefully have some colonies in the morning. Fingers crossed. If not i will cry

Good luck with your 96 constructs!

-Cheers Teagan

[tea-test]

I didn't use the cloning enhancer but i gel purified the insert and used LB medium to grow the cells.

[Teagan27]

Yay... it appears that linearization by PCR has worked a treat. I did an infusion last night. And on one of my plates i had 6 colonies and the other nothing (i had trouble with the gel extraction).. BUT... I did some PCR screening today and guess what? all colonies are positive for the insert. So i imagine that if i had a higher conc. of vector and insert it would have produced more colonies!


Perhaps this is an option you could look into for doing your 96 reactions.


Cheers

[swanny]

Teagan27, on Aug 18 2009, 05:12 PM, said:

Yay... it appears that linearization by PCR has worked a treat. I did an infusion last night. And on one of my plates i had 6 colonies and the other nothing (i had trouble with the gel extraction).. BUT... I did some PCR screening today and guess what? all colonies are positive for the insert. So i imagine that if i had a higher conc. of vector and insert it would have produced more colonies!


Perhaps this is an option you could look into for doing your 96 reactions.


Cheers

I'm just about to try plasmid PCR, as per the Bieniossek paper. They have the advantage that their plasmids are smaller, but that's just a small technical issue.

The problem with InFusion for my 96-reaction expt is the cost of the reaction mix. I have 8 expression vectors (different tag combinations) and I'm cloning 12 homologs. According to the protocol, I have to set up each reaction separately, which will waste lots of vector and insert and cost a bomb! I am trying to get the T4 DNA pol reaction working as it gives me much better flexibility.!

[Teagan27]

swanny, on Aug 19 2009, 03:23 PM, said:

Teagan27, on Aug 18 2009, 05:12 PM, said:

Yay... it appears that linearization by PCR has worked a treat. I did an infusion last night. And on one of my plates i had 6 colonies and the other nothing (i had trouble with the gel extraction).. BUT... I did some PCR screening today and guess what? all colonies are positive for the insert. So i imagine that if i had a higher conc. of vector and insert it would have produced more colonies!


Perhaps this is an option you could look into for doing your 96 reactions.


Cheers

I'm just about to try plasmid PCR, as per the Bieniossek paper. They have the advantage that their plasmids are smaller, but that's just a small technical issue.

The problem with InFusion for my 96-reaction expt is the cost of the reaction mix. I have 8 expression vectors (different tag combinations) and I'm cloning 12 homologs. According to the protocol, I have to set up each reaction separately, which will waste lots of vector and insert and cost a bomb! I am trying to get the T4 DNA pol reaction working as it gives me much better flexibility.!

Ok.. well i have done another infusion with the recommened ratios and tranformed with 1ul of the infusion mix... and only got 20 colonies... at the same time i also ethanol precipitated the remaing infusion (because the infusion enzyme is toxic to the e.coli) and tranformed this in fusion blue cells and got several hundred!  i screened 24 colonies from both plates and 90% of the colonies have the insert. my nightmare is over. The PCR linearization works very well.. but i guess it comes down to cost.

If your interested: I used the Invitrogen platinum taq (its long range and high fidelity) to linearize the vectors... it was very fussy and you must use the magnesium with it (100rxn ~$250). I will let you know if there is any issues with the sequence... But i am thinking that it should all be good!

Cheers.

[swanny]

Great to hear of your success! It's always good to see progress... even if it isn't in your own project!!

Next week, the PCR linearisation begins!

Have a great weekend folks!

[Teagan27]

swanny, on Aug 21 2009, 04:09 PM, said:

Great to hear of your success! It's always good to see progress... even if it isn't in your own project!!

Next week, the PCR linearisation begins!

Have a great weekend folks!

Just to let you know- the sequence came back pretty good. I sequenced 3 of each vector and out of each of them there was only one which had an additional base at the site of ligation.

Hope your PCR linearisation is going well. Thanks for all the help/suggestions.

Cheers, Teagan

[Strong09]

I've been working with the infusion kit for a few months now and had great success.  We've found that performing several double digests on our vector over a few days cut our background down to maybe 1% (18 colonies).  I digested my vector with AfeI/XhoI for 5 hours at a time and then gel purified it.  I then performed another double digest the next day and then phenol/chloroform extracted and precipitated it.  I repeated this 1 more time (3x's digested in total).  I checked for background with vector + ligase and got 18 colonies.  I made sure to gel purify my PCR insert.  Then I used the infusion website to calculate amounts to use in my infusion rxn (2:1 ratio) and diluted the completed reaction with 40 µL of 1X TE buffer.  I then combined 1.5 µL of this dilution with 50 µL of DH5alpha competent cells and plated about half on one amp plate and the remaining half on another amp plate.  Plate one yielded 1000 colonies while plate 2 yielded 769 colonies.  We submitted DNA for sequencing which confirmed the presence of our insert.  Hope this helps with any future projects.

[Andy Lane]

On the subject of removing background from religation of a single-cut vector for SLIC or In-Fusion cloning, has anyone tried using a phosphatase (CIP or SAP) to prevent re-circularization of the vector? None of the protocols seem to mention it, and I can't figure out why. Is the assumption that, in the absence of ligase, re-circularization is irrelevant and that the background must be uncut DNA?

Anyone got any insight? I haven't tried it yet, but it seems like it might help.

Edited by Andy Lane, 02 June 2010 - 02:02 PM.