This month's question from the Molecular Biology Forums (online at molecularbiology.forums.biotechniques.com) comes from the “DNA and General PCR Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&A
How can I improve the quality and yield of my gel purified Internal Transcribed Spacer (ITS)-PCR products? (Thread 26625)
Q I sent 20 purified PCR products for sequencing, but only 4 returned successful sequences even though the concentrations and ratios of the products I sent were good. The personnel in the sequencing core facility suggested that I load the PCR product onto a gel and cut out the bands in order to eliminate some nonspecific products that were interfering with sequencing. But now I get very low yields after purification from the agarose gels.
I extracted my DNA originally using the cetyltrimethylammonium bromide (CTAB) method and ended up with DNA samples that were above 200 ng/µL, but most 260/280 ratios were >2. I did ITS-PCR on the extracted DNA anyway, ran the gels and then cut out the correctly sized bands. After gel purification, my DNA concentrations were extremely low: only 2–5 ng/µL. I concluded that something went wrong during the CTAB extraction because of the high 260/280 ratios. I started again, but extracted the DNA using a Qiagen extraction kit instead. This resulted in good quality, high-concentration DNA with 260/280 ratios of 1.8–2.0. After PCR, the bands looked good in the gels, but I didn't get any better results after gel purification of the PCR bands. The concentrations were low and the 260/280 and 260/230 ratios were poor.
I tried several troubleshooting steps: using 2% and 1% agarose gels of varying thicknesses; running the gels more slowly, ethanol precipitating the extracted DNA to clean it up before PCR, using a Qiagen cleanup kit and an Invitrogen kit, troubleshooting the kits to try to maximize recovery, and minimizing UV exposure of the bands when cutting the gels. I repeated gel purification of this PCR product with these variations more than 10 times with no success. I prefer not to clone the samples if it can be avoided. Can anyone offer any advice?
A What was your PCR reaction volume and how much product did you get? You would need to roughly estimate this by looking at the gel since you cannot use absorbance to quantify with primers and dNTPs in the PCR mix. Did you gel purify the entire PCR reaction?
A 2–5 ng/µL is too low to quantitate accurately by most methods. What were your actual spectrometer readings? Unless you verified the quantity by visualizing the product on a gel, your yield might actually be nothing. 5 ng/µL is too low to sequence at the facility I use.
A I need more information before I can offer advice. Could you answer the questions listed below?
What is the size of the PCR fragment you are trying to gel-purify?
Did you see a big clear band or multiple faint bands when you ran the PCR product on the gel? Is it possible that the band actually contains a number of fragments of the same size?
Did you use a Qiagen Gel Extraction kit to extract the DNA from the gel slices?
How did you determine the DNA concentration? Did you use a NanoDrop or regular 100 µL microcuvette? What is the normal A260 reading range?
A Depending on your PCR conditions, PCR volume, amplicon size and final resuspension volume, 5 ng/µL might be expected. I often run several PCR tubes in parallel to get the amount of DNA I need for direct sequencing.
There was a recent post on Bitesize Bio about making one PCR band from many. You might want to look at it (http://bitesizebio.com/2009/09/14/pcr-rescue). It recommends the band-stab method, which is described in Nucleic Acids Res. 20:4675.
Q Before running the PCR, I extracted the DNA from dry plant material using Qiagen's DNeasy Plant Mini Kit. I used the nanodrop for the genomic DNA, and got concentrations of around 100 ng/µL and A260 range of 1–2. I then diluted the genomic DNA to 1 ng and ran an ITS-PCR on the samples. I cut the bands out of the gel and used the QIAquick Gel Extraction Kit to purify the gel slices. I tried using only 2 µL of the PCR reaction for gel purification and also purified the entire 50-µL reaction.
The fragments vary from 525 to 700 bp, depending on the sample.
Most samples had one large, bright band and 2–3 faint bands.
I used the QIAquick Gel Extraction Kit and also tried using Invitrogen's PureLink Quick Gel Extraction Kit.
I used a Nanodrop spectrophotometer. The average A260 range was 0.1–0.2, with some even lower than 0.1.
A Have you checked each step of the elution protocol to determine where you are losing the product? If you identify where you are losing the DNA, you can easily correct the problem. I would also recommend using a 0.8% agarose gel to more easily elute the DNA from the gel.
Q Since the QIAquick gel extraction kit cleans up the PCR product, wouldn't checking the wash solutions from each step detect the dNTPs and primers from the PCR reaction being washed away, falsely leading me to believe that I am losing my DNA?
A Would you be able to get an Invitrogen TA cloning kit for PCR products? I think New England BioLabs also has a similar kit called UA-cloning. Using one of those kits would eliminate the need for gel purification. You could simply screen Escherichia coli by colony PCR for the desired insert followed by a plasmid mini-prep to collect the DNA for sequencing.
A For fragments of 500–700 bp, you can avoid comigration of fragments of similar sizes by running 2% agarose gels at 70 V. Cut out your DNA bands with minimal amount of agarose, and elute with the QIAquick Gel Extraction kit. Your A260 range is good. You will probably need to compensate for the low yield by pooling multiple samples.
A For sequencing PCR products, I clean them with the QIAquick DNA cleanup kit to remove dNTPs, primers, etc. I always get nice clean reads of up to 900 bases. My suggestion is to skip the gel purification and do a direct cleanup instead, followed by quantification of a 1–2-µL aliquot of the clean DNA on a minigel.
A I started having problems with gel purification after our lab bought a new UV transilluminator. The problems were resolved later when we got a Safe Light light emitting diode (LED) blue light transilluminator (Invitrogen) to use with SYBRsafe DNA gel stain. This led to huge improvements in DNA recovery and subcloning efficiency after gel purification. There is also less background staining for gel images too. If there is a Safe Light available, you might try it to reduce UV damage to your PCR product.
A You might try extracting the DNA from agarose by electroelution or agarase digestion. You could also work on optimizing your PCR so that only one band is produced if that is possible. Then you could use a PCR clean up column, which is more reliable than gel purification.
Q I tried the band-stab method suggested in the Bitesize Bio article and it worked like a charm! Not only was I able to get good concentrations of my bands of interest, but they were clean and had great ratios. I wonder why this simple method is not more well-known. It is much less expensive and time-consuming than cloning.