There's a lot of steps and details here so I will give the overview and if you have specific questions I can give more details on exact quantities/buffer compositions, intermediate steps, and kits used. I apologize this is so long but if anyone could help me I would be extremely grateful; I have been banging my head against the wall for the last month trying to figure out what could possibly be wrong!!
Summary: I am doing three different cloning projects at the moment and they are all are giving me similar unexpected bands when I do a diagnostic digest for verification, even on repeated attempts. It is important to note that with projects 1 and 2, I have had success in the past, but at some point around a month ago, these strange bands started appearing for 100% of my digested clones. It was roughly around this time that I began project 3 (and I saw the same bands even though it is an unrelated project), leading me to speculate that there was some kind of contamination in my solutions that has affected all of my projects. However, I have not been able to isolate where this might be occurring.
1) Generating TA Clones with Promega pGEM T-Easy Kit: Used primers flanking the desired sequence (a promoter of interest, from purified S. Cerevisiae gDNA). Following PCR, this sequence was then gel purified and then ligated into a pGEM T-Easy vector. This vector was transformed into XL-10 gold cells and subjected to blue-white screening on 2XYT-Amp plates with IPTG, X-Gal, and DMF. White colonies were selected, inoculated in liquid media o/n, and plasmids were isolated via Miniprep the following day. These clones were then double digested with NotI-HF and BamHI-HF since these sequences flank the promoter of interest. I have used this method to generate TA clones in the past (roughly 3 months ago) containing my promoter of interest and have seen the small promoter sequence (~300-400 bp) cut from the pGEM vector (~3.0 kB) and also verified by sequencing. The last few attempts, I am only seeing a strong ~2.1 kB band and two smaller, weaker bands at ~3.2 and 4.0 kB (though there are some slight variations between clones, some are slightly lower or higher than by maybe 200 bp). This suggests to me an undigested vector but I am not sure what it might be.
2) Ligating the Promoter Sequence into a New Vector: I digested 2.0 ug of the TA clones with NotI-HF and BamHI-HF to obtain my insert. 2.0 ug of pRS425 (high copy, LEU auxotrophic marker) was digested with the same enzymes to obtain my insertion vector. I ligated them together and then transformed into XL-10 gold cells. Colonies were inoculated o/n and plasmids were isolated via Miniprep the following day. To verify, I digested with the same enzymes. In the past I had obtained positive clones with a frequency of about 30%. I was still seeing unusual band patterns, but there was always at least a few clones that had the expected pattern. For the least few weeks, I have only seen the exact same band pattern each time, either what was described above in 1) or another pattern shifted way up high in the gel (8.0 kB band, 10.0 kB band, and a 10+ kB band beyond the ladder). Note that in the past, I had succeeded with this step and the previous one while attempting with different promoters.
3) Isolating a Plasmid From Yeast Containing a TAP Tag (Plasmid Rescue): I added the DNA sequence for the TAP tag into my plasmid by recombination (from a fragment amplified via PCR) in yeast. I then verified the presence of the tag from selected colonies via Western Blot. These confirmed colonies were restruck and I performed a yeast miniprep. I then transformed this into XL-10 gold cells, inoculated a colony in liquid media, and isolated the plasmid via Miniprep. I digested with Kpn1 which would give me a specific pattern indicating the presence of the DNA sequence of the gene on the plasmid as well as the TAP tag. I have never succeeded in this step and keep seeing the exact same bands as in 1) and 2).
-When I run any of the clones from the three projects uncut, it looks exactly the same as the cut bands (that is, the same mysterious 2.1 kB band with the weaker fragments above it).
-I have tried using a pRS vector as a positive control (for which I know what the band pattern will look like) and it cuts exactly as expected, likely ruling out the enzyme/buffer.
-I performed a "mock miniprep" using all the solutions in an empty microcentrifuge tube. I see the KDS precipitate after neutralization and the KDS pellet as expected. The eluate was transformed into XL-10 gold cells and plated. No colonies grew (suggesting the buffers were not contaminated).
-Most buffers/waters used have been transformed into XL-10 gold cells and plated. No colonies grew (suggesting the buffers were not contaminated).
-Nobody else in the lab using XL-10 gold cells has experienced any problems like this either now or in the last few months when a new stock was made.
-It has been suggested to me that the band I am seeing is undigested pGEM vector which has contaminated something. I selected 6 clones (from project 3) and digested with EcoRI since this site is in pGEM. Some of them produced the same bands as before, some of them produced a single ~3.4 kB band (pGEM is 3.0 kB). Uncut pGEM and cut pGEM (with EcoRI) look the same (3.0 kB).
Okay. I think that's basically everything. If anyone needs any clarification let me know. Thank you SO much for helping out... I am getting really sick of repeating and troubleshooting the same things over and over!
Last edited by SyntonicC
on Jun 14 2012 10:30 am, edited 1 time in total.