Cloning problems and possible contamination

[SyntonicC]

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).

Other Information:
-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!

[relaxin]

1. It seems that your TA cloning failed. Did you screen white colonies by PCR using the same primers that you used to generate the promoter fragment? Are you sure this fragmnet does not contain NotI and BamHI sites? You may try a general blunt-end cloning vector instead of the pGEM T-Easy vector.

2. Did you gel purify the insert before ligating to the pRS425 vector? Did you dephosphorylate the pRS425 vector? Even double-digested vector still need dephosphorylation. Otherwise you may have too many negative clones to deal with.

3. I am not familiar with the Plasmid from Yeast Containing a TAP Tag. Sorry, cannot help.

[SyntonicC]

Thanks for the quick response!

1. It seems that your TA cloning failed. Did you screen white colonies by PCR using the same primers that you used to generate the promoter fragment? Are you sure this fragmnet does not contain NotI and BamHI sites? You may try a general blunt-end cloning vector instead of the pGEM T-Easy vector.

I have not screened the white colonies using the primers - I will make an attempt at doing that and see what I get back. The promoter fragment does not contain NotI or BamHI sites, I have checked all of them. I will take a look and see if we have a blunt-end cloning kit around the lab and if I have better luck with that.

2. Did you gel purify the insert before ligating to the pRS425 vector? Did you dephosphorylate the pRS425 vector? Even double-digested vector still need dephosphorylation. Otherwise you may have too many negative clones to deal with.

Insert was gel purified using a Sigma gel purification kit prior to ligation. I did not dephosphorylate the vector. However, what I have done in the past (many time, successfully before these problems occurred), was set up a vector-only control with the ligation reaction to have a ratio of ligations to vector between my plates. Typically I get 3:1, sometimes 4:1 for ligations to vector and use this as an estimate for how many colonies to pick to increase my chances. I realize this is not ideal so I will give the SAP treatment a try. However, in all the cases where I have done this in the past, I have seen: cut pRS425 for the clones that were vector alone or promoter + pRS425 for the clones for which the ligation reaction worked.

In other words - I usually see the re-ligated vector on my gel if it didn't work. For the last few weeks though, I have been seeing those bands as described in my initial post (and other weird bands too every now and then - where are these bands coming from??)

I would also like to say that for 1) and 2), I have performed the exact same set of steps to generate a library for over 30 promoters into the pRS425 vector with no problems. I am just a little mystified why suddenly out of nowhere things started failing when I haven't changed my methods.

[relaxin]

I have not screened the white colonies using the primers - I will make an attempt at doing that and see what I get back. The promoter fragment does not contain NotI or BamHI sites, I have checked all of them. I will take a look and see if we have a blunt-end cloning kit around the lab and if I have better luck with that.

You do not need a blunt-end cloning kit. Just find a general cloning vector (pUB, pBlueScript) with EcoRV site, cut it with EcoRV, and dephosphorylate. You can also use other site, just fill-in the sticky end and dephosphoyrlate. The PCR fragment should be generated with Pfu polymerase (or other proof-reading polymerase), gel purified, treated with polynucleotide kinase, and purified again with PCR Purification spin column before ligated to the vector.

Insert was gel purified using a Sigma gel purification kit prior to ligation. I did not dephosphorylate the vector. However, what I have done in the past (many time, successfully before these problems occurred), was set up a vector-only control with the ligation reaction to have a ratio of ligations to vector between my plates. Typically I get 3:1, sometimes 4:1 for ligations to vector and use this as an estimate for how many colonies to pick to increase my chances. I realize this is not ideal so I will give the SAP treatment a try. However, in all the cases where I have done this in the past, I have seen: cut pRS425 for the clones that were vector alone or promoter + pRS425 for the clones for which the ligation reaction worked.

In other words - I usually see the re-ligated vector on my gel if it didn't work. For the last few weeks though, I have been seeing those bands as described in my initial post (and other weird bands too every now and then - where are these bands coming from??)

Past performance does not guarantee future success. A simple dephosphorylation step often saves you lots of work in screening for the clones with the correct insert. Lots of weird things can happen during ligation, especially when the insert does not get ligated to vector efficiently.

[SyntonicC]

Thanks Relaxin! I will go ahead and give your suggestions a try and see if it solves any of my problems.

[SyntonicC]

In my lab, we make the Miniprep solutions in-house; one of the other graduate students makes the solutions and has been for a few years now without any problems from anyone. In the past, he has given me a stock of elution buffer (10 mM Tris-HCl, pH 8.5). About a month ago, I took 30 mL from his stocks for my own use.

Well it turns out that I cannot read labels. The stock for the elution buffer is at 1M - I have been using this for all of my elutions. Could this high concentration of Tris affect the purity of the sample in such a way that restriction digest would fail or cause strange things to happen during transformations into XL-10 gold cells?

This is the only solution which is made at a higher stock. All the other Miniprep solutions are to be used at their concentrations off the shelf.

[relaxin]

1 M Tris will affect elution of DNA off the spin column and restriction digestion.

Lesson learned, make your own solutions or use those from coomrcial kits.