Dear Brian,

May be you can check the isolated plasmid DNA (from 1st time transformed colonies) by restriction digestion.  Also ensure that the Kan is added at correct conc. Have you checked the viability of empty competent XL10 (without transformation). All the best.

Were the first transformants not okay or why are you re-doing this? 

In my experience when re-using an old ligation mix stored in the fridge at 4 °C the transformation rate goes way down. Usually frozen ligation mixes reach a similar transformation rate as the fresh ones. 

How did you store your ligation mixture? 

@ Julian thanks for getting back so just to clarify a bit, this experiment was more of a control experiment. My hypothesis was that if the DNA I'm extracting is my target gene contained within the pET-28a plasmid, then it should easily transform again once extracted. An unnecessary step, but bear with me on it.

I think there may be an issue with my clean-up kit. I am using the Promega vector cleanup kit. Has anyone had any issues with this before?

@ Sam I would normally check via sequencing with Eurofins but lately they've been giving me some hassle so I wanted to see if this quick experiment could give some clarification as to whether or not my cells did indeed contain my ligated product.

This cloning procedure has been handed down to me thoroughly optimised. I trust the Kan concentrations are accurate. And these cells have been used prior in transformation procedures, so I don't think they're the issue.

Dear Brian, 

Okay, now I know why you are doing this. I usually do a colony PCR to check for my insert and verify the cloning. For me it works to get a small amount of cells on a pipett tip and suspending it in 50 µL of water. I use only 1 µL of this solution as a template. If you use to much cell matter the PCR won't work for some reason. I check for PCR products on a 1 % agarose gel. 

Since you already extracted the plasmids you could also do a dna restriction and check for the correct pattern via gel electrophoresis. E.g. Eco130I cuts twice, upstream and downstream of the MCS, leaving two pieces of dna. The backbone piece is 5,103 bp big and the part with the MCS ist either 239 bp big without an insert or with your insert about 1,000 bp big. 

Since you have trouble re-transforming with your extracted dna I would agree that there is something wrong. Even if you transform the empty vector you should get transformants. I also use the promega kit for plasmid isolation and I had no troubles yet. Maybe one of the solutions is contaminated or otherwise not okay. Are you sure the ethanol has been added to the washing buffer? Maybe use a new vial of elution buffer as well. 

How high was the initial transformation efficiency [cfu/µg dna]? Did you use the same amount of dna for the second transformation? 

The an other thing I could imagine is a problem with the SOC medium. Was it stored at room temperature? Was it kept in the fridge or freezer until use? Still sterile? 

Colony PCR sounds like a good idea (I'm new to this field so still learning the techniques) I think I'll give that a go. I've cultured one of the same colonies as before overnight last night so I'm going to try an extraction and sequencing again to see if there's any change.

Transformation efficiency was low after ligation: Two colonies per 20 uL ligation product transformed. And no, for the second transformation I used 4uL of a 1:10 dilution of my plasmid. 

The SOC is stored at 4*C and I always check for contaminants before using it. The fact it is still working under more assured conditions (transforming empty pET-28a) makes me think it is fine. However, it was prepared perhaps 1-2 months ago. How long does SOC stay fresh?

Thanks for all your help.

So I grew up another one of the colonies from ligation to see if there was something wrong with the extraction and purification step. All went well, dense cloudy solution showed it was apparent there was plenty of e. coli there. Checked my final purified conc and it's still very low: 18.2ng/uL.

I am going to start again pre ligation to see if I can rectify it somewhere down the line. The nanodrop was only a recent addition to the lab so perhaps it can shine some light as to where the issues are occurring.

I do not prepare my SOC every month so mine is equally fresh. 

Good luck! 

And when you find out what the problem was, please let us know. 

I'd guess the problem might stem from pET28a itself.

In my hands this vector always yields low product amounts when isolating form a 5 mL culture with a mini-prep kit. Its rather a low-copy plasmid, so try to perform an 50 mL culture and isolate with a midi-kit or upscaling your mini-kit. An ethanol precipitation at the end might help to get cleaner plasmid, too.

@ SOC media: I prepared one bottle at the begin of PhD, stored it at 4°C and used it for years without trouble. So I'd say 2 month old SOC is absolutely fine, if it was stored cool. ;D