Could your antibiotic be old? G418 has to be stored in the freezer and in the dark.
If it's ok, then try waiting another 2-3 days or increase concentration further(?).
Also, somewhat unlikely but possible, could your cells be contaminated by a G418 plasmid of some sort? It could happen if you have done many transfections before.
A linearised plasmid should run at the size corresponding to its actual bp length. Uncut or nicked plasmids may run at various sizes, but should be consistent from time to time. Your 2 bands could be uncut and nicked DNA, ie some partial digestion maybe?
My method's pretty similar. I add equimolar amounts of both oligos, but if you dissolve oligos in water, you have to then add STE buffer (I find as high as 90% STE total volume is good). Then heat at 95 for about 15 mins, and allow to cool slowly to room temperature- either use a heating block, and take the metal block off onto the bench, or use a pcr cycler (gradually cool). As far as purification, run on gel and purify. In theory shouldn't matter, as ssDNA will not be incorporated into anything you're working with, such as plasmids
I have very strong doubts you'd be able to see such small fragments no matter what you do. How far into your PCR products are your restriction sites? Enzymes normally need at least 6 bases distnace from recognition site to be present to cut. Also, do you have a transformation control (same plasmid but undigested, no insert) to make sure your cells are still competent?
I agree with hula, it could be something other than dna that you've isolated. Otherwise could be a number of things:
1) Did you have an ethanol wash as the last step of your protocol? If the ethanol wasn't dried off properly your nucleic acid will not dissolve. I normally dry my samples with a pipete tip (capillary action, upside down) and then leave them for a while with lids open (cover with tissue or something) on bench or ona heating block (not too hot). With the samples you alredy have you can do an ethanol precipitation and redissolve
2) Your dna might be very concentrated and so reached the extent that it'll dissolve, what's the concentration? Try adding more buffer.
3) Leave your dna at 4C overnight to allow to dissolve, genomic dna takes a while to dissolve
Hope this helps!
I do apologise, but I believe some of the answers above are incorrect.
Here's my version, choose whichever you like:
2= (not 100%sure, but A sounds closer to the truth)
8= A (no doubt about this one!)
I tried Calc Phos, but it didn't seem to be particularly good. A similar methodology, but using Dextran sulfate normally works better, but there are problems with toxicity, so you got to watch it. Lipofectamine seems quite good, but you might still try smth different, like Fugene6 or DOTAP, there are tons of different reagents on the market now.
I add pen/strep to all my cultures, including transfections. You're better off in most cases with a lower transfection efficiency than no cells at all due to contamination. Some transfection reagents/methods work better with some cells and not others, so I suggest use pen/strep but maybe look at other transfection methods.
If your contamination occurs only in 4 out of 240 reactions this is a rare event, you must be getting contaminants in some of your buffers or other reagents, but the concentration of contaminating DNA must be low, so that when you aliquot your samples you don't get enough template in many reactions. Get fresh buffers/ aliquot, and discard as soon as contamination apperas.
As for your actin problem- it is not actually as good a housekeeping gene as some people believe. I don't know what your system is, but your treatment may actually influence actin, try a different control, such as GAPDH, PBDG or even 18S rRNA.
I have never tried Zymogen kits, but Qiaquick works well normally. A few possible problem areas:
1) How big is your PCR product? Has to fall within the limits specified in the kit
2) Qiaquick is the gel kit, right? So you actually have to cut out your product. How intense was your band of interest? Maybe your PCR wasn't particularly good
3) Check the pH of your elution buffer, or (especially!) water, DNA will not be eluted if pH is too low, has to be slightly basic. (MQ water is not nevessarily neutral)
4) How old is your isopropanol? It's hydrophilic, so if it gets opened too often, it will be "diluted", so your concentrations will change. Get a new batch
Hope this helps!