Cloning purified PCR product into cells - (Jan/21/2015 )

I am currently trying to sequence the end of a gene. We have 75% of it. I use a designed primer as well as a 3' adapter primer that should amplify from the polyA tail. My PCR results show positive results on an agarose gel and I do have DNA after purifying the PCR however when I transfect the purified DNA into bacteria, nothing grows. I have a positive control with this and it always grows. 

The only thing I cant think of is that my PCR product is only single stranded DNA. How do I get around this? What am I missing?

Spacepirate, I am sure you know that transfecting PCR product of your gene will not grow bacterial colonies; one would need to insert that product into a plasmid first. But since you haven't mentioned that step, and the fact that you think your PCR product is a single stranded DNA, makes me concerned about the basics here. 

You need to ligate your PCR product to a cloning vector in order to transform your cells.   The PCR fragment on it's own won't work, it doesn't have the required material for replication and/or expression. 

You should choose the vector according to your downstream application, if you just want to sequence, any of the T-cloning vectors should work pGEM-T, Topo....  

Have a look here for the basics:   http://en.wikipedia.org/wiki/Molecular_cloning

If you google Molecular Cloning you'll get a lot of information.  Also, here's the cloning bible:  http://www.molecularcloning.com/

This should help too:  https://www.neb.com/~/media/NebUs/Files/Brochures/Cloning_Guide_1113.pdf

I suppose I wasnt as thorough in writing my question as I should have been. My PCR product is purified and ligated into a cloning vector then grown in bacteria. My problem is nothing is growing. I run a positive control as well with a gene we know can and has cloned into bacteria, and it does grow on the plates very well.

What I'm questioning, is why I would have such a positive result on a gel, and there is dsDNA present (according to the spectrophotometer), and still nothing grows once its been cloned into a vector and grown on a plate? (I do the cloning with my mystery gene as well as my positive control at the same time, so they are subject to the same protocol.)

I am glad that you are well versed with the technique. However, troubleshooting problems in cloning of PCR products is a long subject. So I suggest you talk with a colleague, read up some troubleshooting guidelines on this forum previous posts and here, and then if you still have troubles, write to us. Seriously, this is not something I can write down 10 points and problem will be solved.

spacepirate,

just some points.

First, you can hardly get a ssDNA from PCR, that is not how PCR work. If you work on mRNA, and reverse transcribe, then you have a mRNA-cDNA duplex, that can be used (the cDNA part) as a template for a PCR.

RT products are hardly visible on gel (since the is just one cDNA for one mRNA molecule), so I would say you did PCR on your RT product, and that makes a maaaaany dsDNA fragments inbetween your primers. So you don't have a ssDNA.
(Also spectrophotometer can't tell you anything about double-strandedness of your product, it measures bunch of nucleotides the same as RNA or ssDNA or dsDNA, long fragments, short fragments.. it just measures basic "chemical" stuff. It can't tell you if it's ds or ss or even degraded or nice PCR product. Only when you KNOW you have a nice PCR product there, and nothing else, only then int can tell you HOW MUCH of it you have. But since you saw your product on gel fine, you have some amount od dsDNA there. But spectorphotometer can say nothing yout its quality on its own.)

Second, you say it ligated but doesn't grow. Since growth is a kind of a prove of ligation, you actually can't be sure, that you successfully ligated your PCR insert.

Your positive control can be pretty different. Your comment suggest that the PC is a gene cloned same way as the fragment you are trying now, are you recreating the whole ligation together with your problematic gene? If not, it's good to do it, since there may be something faulty with your ligase.
Classic ligation is usually a "multiple-try" adventure when you need to try different insert:vector ratios to get the one that works. But for PCR products there are many available kits that are easy as pie (TA cloning for example). Can you provide more information on what do you use for ligation and what modifications you tried?

Third, lets say you prepared the positive control completely the same (including similar length) and control vectors grow, but your darn gene does not. Bacteria doesn't like all the stuff you put in them the same, especially when the express it. Sometimes it happens, that the insert or combination of vecotr and insert (again, describing more details about what vector you use, if it has bacterial promoter and so) is toxic for cells or recombines or something. But without providing additional information about.. generaly anything you did, we can't help you here.

Each of these three comments can be summed up as three possible problems: problem one - toxic or instable insert, problem two - failed ligation, problem three - you actually have no idea what you are doing. 
Not necessarily in this order.