The Issue
The most important aspect in the successful use of pin tools is to start with clean pins. We strongly recommend that you use our specially developed pin cleaning solution VP 110A at the beginning and end of each day to keep the pins clean. It only takes a few seconds of treatment in the VP 110 solution to remove built compounds, proteins, and finger oil. Just dip the pins in the diluted VP 110A solution for 10 to 30 seconds followed by blotting and triple dipping* in a distilled water bath and an alcohol (isopropanol, ethanol, or methanol) bath to clean a pin tool. Keep your pin tool delivering consistent volumes every time with this simple maintenance.
Our most common Technical Service question for Pin Tools involves poor precision or accuracy. In virtually every case the problem goes away when the customer uses our VP 110A Pin Cleaning Solution and proper pin cleaning technique. It is a simple and inexpensive way to maintain consistent results.
Potential Problems and Solutions
DNA and High Surface Tension of Spotting Solutions
Spotting DNA onto membranes or glass slides can be problematic because the spotting solutions often have a high surface tension which results in variable volume hanging drops and therefore, variable spot diameters. We first noticed that distilled water has a very high surface tension and when used with dye (food coloring) to make practice arrays, this mixture produced highly irregular spot sizes. We found that the irregular spot size can be eliminated by adding a detergent such as Tween 20, Sarkosyl, proteins, carbohydrates, or carrier DNA to the solution. However, there are many protocols where it is not possible to add these surface tension lowering compounds to the solution.
An alternative way to address the problem of high surface tension liquids is to clean the pins with our VP 110A solution (diluted before use) which cleans the pin of proteins and other residues and changes the surface tension of the pin thus compensating for the high surface tension of the liquid. Dipping the pins into this solution briefly cleans the pins and changes the surface tension of the pin. The pins are washed in water and ethanol to remove any residual solution and used right away. This pin cleaning treatment results in very uniform dispense volumes.
Improper Cleaning and Oils
Pins will not deliver a consistent volume if they are not cleaned after use, or if they are contaminated by finger oil. A single 10 second treatment with the V&P Pin Cleaning Solution removes built-up protein and oils and dramatically improves delivery consistency and reduces CVs.
Pin Slots
It is possible that the high surface tension buffer may not wick and fill slot pins uniformly. Usually, this is due to the slot not being cleaned properly. This will result in variable spot sizes and transfer volumes. This can be overcome by cleaning the slot pins with the V&P Pin Cleaning Solution as above. The ability to have slots filled by capillary action is highly desirable when working with small volumes of DNA or PCR reaction products. This wicking ability allows you to recover even the “dregs” at the bottom of a PCR well.
Before V&P Cleaning Treatment
After V&P Cleaning Treatment
The 2ul blots above were made with a VP 408S2 Slot Pin Replicator using distilled water and 5% red food coloring on Hybond-N Nylon membrane (Amersham Pharmacia, P/N RPN 1510 N) illustrates the problem.
Once the V&P Pin Cleaning Solution has cleaned the slots and modified the surface tension of the pin, the modified surface tension remains even after 15 rinses in 100% ETOH or distilled water. It even remains on the pins after treatment with 5% bleach. Thus, the replicator pins can be used for many blots or transfers without re-treatment. We recommend that the V&P Pin Cleaning Solution be diluted 1 to 5 and used at the beginning of each spotting session. After dilution and use, the V&P Pin Cleaning Solution can be stored and used again and again.
When it comes to keeping pins clean, the type of blotting paper used is also very important. Most blotting paper contains lint which can stick to the pin tips and slots. This will interfere with the pickup of liquid on the pins. We strongly recommend using a lint-free blotting paper. We also have a super absorbent polypropylene pad in a tray covered by lint-free paper (VP 540DB) that will hold up to 27 ml of liquid. This means you can make 100-10,000 blots without changing the blotting paper.
Flame Treatment
You can significantly lower the surface tension on the pin by flaming** from ETOH or isopropanol. This also results in very uniform drop size and slot filling. This is convenient if you can accommodate flaming in your replicating or spotting procedure.
* Triple dipping entails removing the pins from the bath and immediately dipping back in 3 times. This gives a thorough washing of the pins.
** Flaming just entails igniting the alcohol on the pin and NOT heating the pins directly in a Bunsen burner which can damage the corrosion-resistant properties of the stainless-steel pins.