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Serious cloning issues - gel extractions?

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Serious cloning issues - gel extractions?

Postby TaintedCherub » Thu Oct 15, 2009 11:27 am

Dear all,

For several months now I have been attempting what is on paper a fairly simple subcloning procedure, without any success, and I would really appreciate any advice. I have no previous cloning experience before this. I have tried quite a few approaches now and will try to cut a long story short.

I am isolating an insert by BamHI digestion from the donor construct, BamHI digesting the recipient vector (one cut), dephosphorylating the digested vector and then trying to ligate the insert and linear vector via the BamHI sticky ends and transform the resulting construct into supercompetent cells.

The insert and the linear vector are both digesting as expected, giving the correct bands at the correct weights on gels. I am gel extracting the insert and linear vector, then pooling the extractions, EtOH precipitating, ligating and transforming. This is where the problems begin. The transformations yield no colonies at all for ligations, but plenty for positive control uncut vector. I have also tried a mock ligation with uncut vector. This transforms fine, so the ligation mix is not toxic to the cells.

I have tried to simply ligate BamHI digested, gel extracted linear vector back to itself. 100 ng DNA, 1 U ligase for 10, 60 and 120 minutes at 37oC, using fresh ligase. None of these reactions resulted in any colonies, despite uncut vector producing a near-lawn. I have been using an estimated 10 ng DNA in transformations.

The only hint I have is that, following gel extraction and precipitation, there is always a peak in DNA absorbance at 220-230 nm, resulting in 260/230 ratios sometimes below 0.5 (260/280 is usually 1.6-1.8). Also, during the latest precipitation, I noticed white flakes forming that were insoluble in water, which I span down and removed.

It seems like something is carrying over from the gel extraction that is either inhibiting ligase or killing my supercompetent cells. Possibly guanidium salts or agarose contaminants? I have now tried both Qiagen and Invitrogen gel extraction kits (silica column types) with identical results.

Does anyone have any experience with similar problems? I have a couple more controls to try, most importantly gel extracting, mock ligating and transforming uncut vector, but by this point I am fairly certain it is a problem with gel extraction using these kits. The next step is to try using DEAE membrane and salt elution.

Time is also an issue, as I only have 6 months left in my PhD project, and I have been grappling with this problem since April. After this, I still have to do some site directed mutagenesis and create stable transfections before I can even start my final year's data collection, so I am feeling under a lot of pressure. Help would be great, anyway! Thanks.
Last edited by TaintedCherub on Thu Oct 15, 2009 11:39 am, edited 3 times in total.
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Postby JackBean » Thu Oct 15, 2009 11:37 am

You are doing ligation at 37°C? As I remeber, I have always used like 16-18°C O/N.

Did you check on gel, whether the ligation really works? Let's say, in one row have ligated product, in another unligeted mixture, in another ligated product cut with BamHI and in other ligated product cut by some other RE(s)?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby TaintedCherub » Thu Oct 15, 2009 11:41 am

JackBean wrote:You are doing ligation at 37°C? As I remeber, I have always used like 16-18°C O/N.

Did you check on gel, whether the ligation really works? Let's say, in one row have ligated product, in another unligeted mixture, in another ligated product cut with BamHI and in other ligated product cut by some other RE(s)?


Hi, thanks for the quick reply.

I have tried it at 16oC overnight, but that didn't work either (including cut, not dephosphorylated vector).

I am actually in the process of running a gel now to check the ligations, I can let you know the results later.
Last edited by TaintedCherub on Thu Oct 15, 2009 11:44 am, edited 1 time in total.
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Postby JackBean » Thu Oct 15, 2009 11:43 am

Didn't work the transformation or ligation? I suggest to check out the ligation product and let us know tomorow ;)
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Postby JackBean » Thu Oct 15, 2009 12:32 pm

Actually, if are you doing the gel extraction, I guess, you have run the agarose electrophoresis, didn't you? :lol: So, you should know, what does it look like.

BTW why don't you use only the lgation reaction for transformation? Without the gel extraction ;)
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Re: Serious cloning issues - gel extractions?

Postby TaintedCherub » Thu Oct 15, 2009 3:03 pm

If something is carrying over from the gel extraction I guess it could be scuppering either the ligation or the transformation. Hopefully the gel should confirm whether the ligation has worked! I just started it running overnight, so should know tomorrow morning.
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Postby JackBean » Thu Oct 15, 2009 10:18 pm

What is the order of steps you are doing? Where is the gel extraction anyway?
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Postby kk » Fri Oct 16, 2009 10:16 am

Hello, nice (sad) to see I am not the only one unable to do the simplest cloning process. I am also a beginner.

My situation is almost exatly the same, except I use BamHI and XhoI. Can I ask you, how come that you are using BamHI only? I thought multiple cloning sites (MCS) are designed to have mostly unique restriction sites.

Basic question, but are you sure you are using the right antibiotics at the right concentration?

My 260/230 is also very low for this batch that does not want to work. I did not check it for the previous cloning I did and succeeded.

I was just told today that I should wait longer with my plates before I throw them out in the morning - maybe colonies appear in just a few hours later.
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Postby kk » Fri Oct 16, 2009 11:35 am

I was told by a PhD student that even the way you put your LB-plates in the incubator could matter: if they aren't upside down they could dry out. I think that is too much, it can't be that critical over one night...
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Postby JackBean » Fri Oct 16, 2009 12:18 pm

They shouldn't dry up, but conversely the evaporating water condensates on the lid and if it is on the top, then the water can drop back down and mix your colonies ;)

Yeah, MCS have unique restriction sites, but they can be twice in there, or, more importantly, you can insert your DNA through only one RE place. That is kind of tricky, because it can be inserted in any way, so you have to check afterwards.
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Postby MrMistery » Fri Oct 16, 2009 2:56 pm

Welcome to cloning hell buddy, there's plenty of us in here :-)
Anyway, I don't think the gel extraction is your problem, especially since you used two different kits. It may be your elution though, are you eluting in EB buffer or water? If your water is too acidic, then all the DNA will remain on your spin column instead of eluting. Just run 3 uL of the gel extracted DNA on an agarose gel and see if there's anything there (though you said you did absorbances, so there should be).
Asides from that, all I can think off the top of my head are the usual stuff (get new T4 Lig Buffer, make sure the buffer and enzyme are from the same manufacturer etc). I'll think about it, let us know what you get though
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Re: Serious cloning issues - gel extractions?

Postby TaintedCherub » Wed Oct 21, 2009 3:46 pm

Thanks for all your replies, and sorry for the delay in updating!

I have run a couple of new gels, detailed at the bottom of this post. To try and answer some of your questions:

I am using BamHI because the insert has a single BamHI site at each end, while the recipient vector is cut once in the MCS by BamHI. The sticky ends will be compatible, but I will have to screen for orientation as JackBean commented, as the insert could go in either way round.

I have checked the antibiotics (Kanamycin; 30 ug/ml) and my undigested empty vector controls are transforming fine. I have done quite a few successful transformations so I doubt that this is the issue, but it's a good thought, cheers. I always incubate plates upside down and leave them an extra few hours just in case.

I have been eluting in water at 60oC for 10 minutes and nanodrop readings suggest reasonable recovery of DNA (40-60%), but looking at my latest gels below this could well be an issue! I will use TE buffer from now on. I have been using injection grade water, which I pHed once and found to be slightly alkaline, about pH 8 I think.

The T4 ligase was my first thought. I am on my third batch now. The buffer is frozen in aliquots so no freeze/thaw cycles, I heard that ATP is fairly unstable in that situation.

JackBean, the steps I have been using are as follows: Maxiprep, precipitation, BamHI digestion, CIP dephosphorylation of vector, immediate gel extraction of insert and linearised recipient vector, pooling and EtOH precipitation, ligation, transformation. My advisor commented this week that it might be worth omitting the gel extraction and just seeing what happens, so I plan to try that soon.

Here are the latest gels:

http://crayfish.zenfolio.com/p743268062 ... #h1c2df31f

Circularised vector ligation reactions run out on a gel. No DNA was detectable. According to the Nanodrop there should have been 75 ng per lane.

http://crayfish.zenfolio.com/p743268062 ... #h183cc9d1

Gel extracted linear vector and insert run out on a gel. Insert was present, though slightly faint, but linear vector was only barely detectable at the highest exposure. Again, according to the Nanodrop there was 200-250 ng DNA per lane. Looking at the high exposure image on the right, a faint band can be seen in the linear vector lane, with a smear below it. Does this indicate degradation? The band is at too high a molecular weight so might be an artefact of some kind. On the other hand the insert is also running about 500 bp too high according to my linear ladder.

If the gel extracted vector is degrading for some reason then this might explain the discrepancy between the nanodrop readings and the amount of DNA observed on the gel, as small fragments / single nucleotides still absorb at 260 nm. Odd that I would only get degradation in one sample though!

Interesting to hear that you are also getting a low 260/230 ratio... have you tried running the DNA back onto a gel to check it's really there kk? Is that using a silica spin kit after gel extraction?

I will try my advisor's suggestion of just not doing gel extraction next, unless anyone has a better idea :)

Apologies for the information overload!!
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