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"ladder" ligation result - cloning is the bane of my existance (Oct/26/2008 )

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"...then add 1U Alkaline phospatase and cook for an hour more."

Man, that's way high. We use 1/10th of that amount, and incubate at 37C for 45 seconds to one minute. Anything more, and we wind up with vector that has damaged ends and no clones.

-HomeBrew-

Hi folks,
I re-did the ligation with vector not treated with AP. I then transformed. I had one colony, which had empty vector, as a result. Below are two images of my ligation reactions. The first is with AP treated vector. The second is with not dephosphorylated vector. I don't really see a difference. What do you guys think the images indicate?

Thanks to everyone who replied! I'm considering re-ordering my primers and changing restriction enzymes.

With AP:
ladder, no insert control, 1:3, 1:5
[attachment=5516:ligation10_8.jpg]


Without AP:
ladder, 1:5, no insert control, no ligase control
[attachment=5517:ligation10_30b.jpg]

-d.g.alina-

I would say that if you have enough DNA to see on a gel, you have too much, unless your reaction is 50 ul. Too much vector leads to intermolecular interactions. That's not just me either, go and check Maniatis, or the later editions.

-swanny-

QUOTE (swanny @ Nov 4 2008, 11:20 PM)
I would say that if you have enough DNA to see on a gel, you have too much, unless your reaction is 50 ul. Too much vector leads to intermolecular interactions. That's not just me either, go and check Maniatis, or the later editions.


I did a 20ul reaction with 40ng vector and 100ng insert. I can see 20ng easily on my gels with a long exposure.

-d.g.alina-

QUOTE (d.g.alina @ Nov 5 2008, 03:12 AM)
Hi folks,
I re-did the ligation with vector not treated with AP. I then transformed. I had one colony, which had empty vector, as a result. Below are two images of my ligation reactions. The first is with AP treated vector. The second is with not dephosphorylated vector. I don't really see a difference. What do you guys think the images indicate?

Thanks to everyone who replied! I'm considering re-ordering my primers and changing restriction enzymes.

With AP:
ladder, no insert control, 1:3, 1:5
[attachment=5516:ligation10_8.jpg]


Without AP:
ladder, 1:5, no insert control, no ligase control
[attachment=5517:ligation10_30b.jpg]

OK, I just had a chance to look at the gels again without interruptions and with a clear head.
I would say your "no AP" samples show that both insert and vector are well-digested. The laddering looks good. You appear to have one band that could be vector plus insert (just under 4kb, fourth band from the bottom of the 1:5 lane).
The AP-treated gel is harder to tell, because the bands are quite faint, but I would say that one is more successful. A quick play on Photoshop suggests both ligations have been successful, but the 1:5 has been better. The 1:5 lane shows very little remaining insert (good), less vector than the other lanes (also good) and a good band of DNA about the right size for a successful ligation (once again, good). The problem might lay in the transformation.

What is the transformation efficiency of the cells? How long do you let the cells recover after the heat-shock? I'd suggest you give them at least an hour before plating them out. Also, I would plate out more of the transformation mix, even if you do it all on a couple of plates.

I think you are very close to cracking this one!

-swanny-

QUOTE (swanny @ Nov 5 2008, 09:07 PM)
QUOTE (d.g.alina @ Nov 5 2008, 03:12 AM)
Hi folks,
I re-did the ligation with vector not treated with AP. I then transformed. I had one colony, which had empty vector, as a result. Below are two images of my ligation reactions. The first is with AP treated vector. The second is with not dephosphorylated vector. I don't really see a difference. What do you guys think the images indicate?

Thanks to everyone who replied! I'm considering re-ordering my primers and changing restriction enzymes.

With AP:
ladder, no insert control, 1:3, 1:5
[attachment=5516:ligation10_8.jpg]


Without AP:
ladder, 1:5, no insert control, no ligase control
[attachment=5517:ligation10_30b.jpg]

OK, I just had a chance to look at the gels again without interruptions and with a clear head.
I would say your "no AP" samples show that both insert and vector are well-digested. The laddering looks good. You appear to have one band that could be vector plus insert (just under 4kb, fourth band from the bottom of the 1:5 lane).
The AP-treated gel is harder to tell, because the bands are quite faint, but I would say that one is more successful. A quick play on Photoshop suggests both ligations have been successful, but the 1:5 has been better. The 1:5 lane shows very little remaining insert (good), less vector than the other lanes (also good) and a good band of DNA about the right size for a successful ligation (once again, good). The problem might lay in the transformation.

What is the transformation efficiency of the cells? How long do you let the cells recover after the heat-shock? I'd suggest you give them at least an hour before plating them out. Also, I would plate out more of the transformation mix, even if you do it all on a couple of plates.

I think you are very close to cracking this one!


That's great to hear!

I think you're right that the cells are (in the very least) part of the problem. I am transforming into HT115 cells, which are relatively slow-growing. They are the bacteria we feed to c. elegans in order for them to uptake the RNAi.
I get random positive control failure with my cells are my protocol. I've tried DHh5alpha cells, too.
I am following the only protocol for HT115 cells I have ever seen:

Thaw on ice until no crystals are left (~30 min).
Aliquot 100ul into round-bottom tube.
Add 1-5 ul ligation reaction (I generally don't clean it up, though I tried using 1ul after a column kit and it still didn't work)
Incubate on ice 30min
Heat-shock at 37deg. for 1-3 min (I tried varying this time... no change).
Add 900-1000ul SOC/LB (I used SOC until recently, when I ran out... doesn't seem to make a difference for the positive control)
Shake at 37 deg. for 1 hr+
Add and spread 20ul of transformation on one half of the plate. Add 100ul to the other half.

Like I said, I get really unreliable results from this transformation.

-d.g.alina-

QUOTE (d.g.alina @ Nov 7 2008, 12:43 AM)
QUOTE (swanny @ Nov 5 2008, 09:07 PM)
QUOTE (d.g.alina @ Nov 5 2008, 03:12 AM)
Hi folks,
I re-did the ligation with vector not treated with AP. I then transformed. I had one colony, which had empty vector, as a result. Below are two images of my ligation reactions. The first is with AP treated vector. The second is with not dephosphorylated vector. I don't really see a difference. What do you guys think the images indicate?

Thanks to everyone who replied! I'm considering re-ordering my primers and changing restriction enzymes.

With AP:
ladder, no insert control, 1:3, 1:5
[attachment=5516:ligation10_8.jpg]


Without AP:
ladder, 1:5, no insert control, no ligase control
[attachment=5517:ligation10_30b.jpg]

OK, I just had a chance to look at the gels again without interruptions and with a clear head.
I would say your "no AP" samples show that both insert and vector are well-digested. The laddering looks good. You appear to have one band that could be vector plus insert (just under 4kb, fourth band from the bottom of the 1:5 lane).
The AP-treated gel is harder to tell, because the bands are quite faint, but I would say that one is more successful. A quick play on Photoshop suggests both ligations have been successful, but the 1:5 has been better. The 1:5 lane shows very little remaining insert (good), less vector than the other lanes (also good) and a good band of DNA about the right size for a successful ligation (once again, good). The problem might lay in the transformation.

What is the transformation efficiency of the cells? How long do you let the cells recover after the heat-shock? I'd suggest you give them at least an hour before plating them out. Also, I would plate out more of the transformation mix, even if you do it all on a couple of plates.

I think you are very close to cracking this one!


That's great to hear!

I think you're right that the cells are (in the very least) part of the problem. I am transforming into HT115 cells, which are relatively slow-growing. They are the bacteria we feed to c. elegans in order for them to uptake the RNAi.
I get random positive control failure with my cells are my protocol. I've tried DHh5alpha cells, too.
I am following the only protocol for HT115 cells I have ever seen:

Thaw on ice until no crystals are left (~30 min).
Aliquot 100ul into round-bottom tube.
Add 1-5 ul ligation reaction (I generally don't clean it up, though I tried using 1ul after a column kit and it still didn't work)
Incubate on ice 30min
Heat-shock at 37deg. for 1-3 min (I tried varying this time... no change).
Add 900-1000ul SOC/LB (I used SOC until recently, when I ran out... doesn't seem to make a difference for the positive control)
Shake at 37 deg. for 1 hr+
Add and spread 20ul of transformation on one half of the plate. Add 100ul to the other half.

Like I said, I get really unreliable results from this transformation.

Good luck with it all. Try transforming the cells with something like pUC, working out how much DNA you add to the cells, how much media you add, and how much you plate out to calculate the transformation efficiency per ug of DNA. It might also be helpful to do the experiment with the plasmid backbone as well, just to see if there's something strange about the plasmid itself.

-swanny-

Got it! I was finally able to see colonies when transforming DH5alpha cells (the home-made HT115 situation may be hopeless...) and plating 500ul of undiluted transformed cells. I still can't believe I only had 4 colonies after I dumped all that onto the plate (3/4 colonies had the insert!)

Thanks again to everyone who responded! Much appreciated!

Now to somehow get my vector into HT115 cells so I can feed it to my worms...

-d.g.alina-

QUOTE (d.g.alina @ Nov 9 2008, 01:09 PM)
Got it! I was finally able to see colonies when transforming DH5alpha cells (the home-made HT115 situation may be hopeless...) and plating 500ul of undiluted transformed cells. I still can't believe I only had 4 colonies after I dumped all that onto the plate (3/4 colonies had the insert!)

Thanks again to everyone who responded! Much appreciated!

Now to somehow get my vector into HT115 cells so I can feed it to my worms...

I presume there aren't any companies out there that make competent HT115s...

At least now you can confirm sequences etc, so when you transform into HT115, you'll know that you're adding what you think you're adding!

-swanny-

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