Frequently Asked Questions

     
         
 

Q: How do I determine which pin (coated or uncoated, slotted or solid, 33 mm or 50 mm long, .229 mm to 1.58 mm diameter) is best for my application? Answer

Q: Do you have any scientific references that cite your Replicators and list procedures? Answer

Q: What is the best way to clean Solid, Grooved and Slot Replicator pins if they become dirty? Answer

Q: My slot pins are not wicking up the solution or the slots are not filling all the way up.  My DNA is only 1 ng/ul and is in a buffer that I cannot add surfactant to (detergent, protein, carbohydrate or carrier DNA or RNA).  Answer

Q: The size of my solid Replicator pin spots on membrane are irregular.  My DNA is only 1 ng/ul and is in a buffer that I can not add surfactant to (detergent, protein, carbohydrate or carrier DNA or RNA).  Answer

Q: How do I test the Replicators prior to use? Answer

Q: What determines the volume of liquid transferred by the Replicator pins? Answer

Q: How can I transfer larger volumes using pins? Answer

Q: What is the accuracy of delivery using slot pins? Answer

Q: How can I do quantitative studies on high density arrays made on membranes. Answer

Q: When using the Magnetic Tumble Stirrer, how fast should the stirrers tumble? Answer

Q: Will the stainless steel stirrers react with my reagents? Answer

Q: How should I practice making arrays with Replicators and the registration devices? Answer

Q: When making arrays with the MULTI-PRINT™ should I use blotting paper under the membrane? Answer

Update on liquid transfers

 
 
Q: What is the best way to clean Solid, Grooved and Slot Replicator pins if they become dirty?

A: Although the pins should never become dirty if they are cared for properly during regular use, they can be easily cleaned by sonication for ~20 seconds in a bath that contains 1% Micro 90 cleaning solution.  Micro 90 can be purchased from International Products Corp. Phone (609) 386-8770.

Cole-Parmer Instrument Company (800) 323-4340 sells an inexpensive ultrasonic cleaner, model P-08857-02 that is of the ideal size for cleaning all replicators.

The next best way is to clean the pins with Ivory dishwashing soap and the VP 425 brush.  Once the pins are clean we recommend a final dip in the VP 110 Pin Cleaning Solution.  (TOP)

Q: My slot pins are not wicking up the solution or the slots are not filling all the way up.  My DNA is only 1 ng/ul and is in a buffer that I cannot add surfactant to (detergent, protein, carbohydrate or carrier DNA or RNA). 

A: Just water or NaOH without surfactants is very hydrophobic and will not fill the slots by capillary action.  Dipping the pins in ETOH and flaming them prior to picking up the solution changes the surface tension factor on the pin and allows the slots to fill.  If it is not possible to flame your pins, then treat them once in our new pin Pin Cleaning Solution VP 110.  See the V&P Pin Cleaning Solution page for more information about how this cleans the pin and changes the surface tension on the pin slot.  

Irregular spots can also be caused by lint in the slots from paper towels used to blot the Replicator between cleaning baths. This can be prevented by placing a lint free blotting paper (VP 522) over the paper towel.  (TOP)

Q: The size of my solid Replicator pin spots on membrane are irregular.  My DNA is only 1 ng/ul and is in a buffer that I can not add surfactant to (detergent, protein, carbohydrate or carrier DNA or RNA).

A: Just water or NaOH without surfactants is very hydrophobic and will form irregular sized hanging drops on solid Replicator pins.  Dipping the pins in ETOH and flaming them prior to spotting changes the surface tension factor on the pin and gives much more uniform spot sizes.  If it is not possible to flame your pins then treat your slot pins once in our new Pin Cleaning Solution VP 110.  See the V&P Pin Cleaning Solution page for more information about how this cleans the pin and changes the surface tension on the pin.  (TOP)

Q: How do I test the Replicators prior to use?

A: Make an aqueous solution containing the buffer or media you will be transferring.  Add to that solution the substance (DNA, RNA, protein, carbohydrate, surfactant, etc.) you wish to test in approximately the concentration it will represent in your actual solution.  If that is not possible use a 1% solution of Bovine Serum albumin.  Add several drops of blue food coloring to this solution.  It is very important that a substance (DNA, RNA, protein, carbohydrate or surfactant) be added to the water as water has a very high surface tension and just water with dye will result in erratic volume transfers.  If you can not add surfactants to your buffer, then change the surface tension of the pins by by dipping in ETOH and flaming prior to dipping in the test solution.  The surface tension on the pins can also be changed by cleaning the pins in our new V&P Pin Cleaning Solution (VP 110).  See the V&P Pin Cleaning Solution page for more information about how this cleans and changes the surface tension on the pin.  (TOP)
A2: We provide a complete protocol which will help you to determine the volume of your liquid that is transferred.  See technical note 65.

Q: What determines the volume of liquid transferred by the Replicator pins?

A: A solid Replicator pin transfers liquid on two surfaces: a small hanging drop forms on the tip of the pin, and secondly, on the sides of the pin.  The volume of liquid transferred depends upon the diameter of the pin, the chamfer of the tip, the flat spot, the speed that the pins are removed from the liquid, the surface tension of the liquid, the surface tension of the pin, the depth to which the pin is wetted, the diameter of the well the pin is inserted into, and the distance between the pin and the well wall. Although each of these factors may make small changes in the volume of liquid transferred, we have found that the pin to pin reproducibility is better than 5%.

One of the variables that you can easily control is the speed with which you remove the pins from the liquid - the slower the removal, the smaller the drop size on both surfaces.  You can also add surfactants, proteins or solvents to the liquid or change the pH to change the surface tension forces.

Changing the surface tension on the pin can be done in several ways: 1. dipping the pin in ETOH and flaming, 2. cleaning the pin with the V&P Pin Cleaning Solution (VP 110) or 3. washing in a detergent such as Ivory dishwashing soap.

The LIBRARY COPIER™ (VP 381) is used to keep all the pins in the middle of the wells and to keep from knocking off the hanging drop.  Although there are several different variables, we have determined that with nearly all the biological specimens that are transferred, these variables are negligible and thus the volume transferred remains the same for all the pins even when significant differences in protein concentration exist.  Furthermore there is excellent reproducibility between different Replicator stampings or blots.

The liquid transferred to membranes or agar comes only from the hanging drop on the tip of the pin so by changing the chamfered spot diameter and/or the pin diameter and shape we can offer a variety of custom delivery volumes.  Contact us with your special needs.  (TOP)

 


Update on Liquid Transfers

     
  Delivery volumes for floating pins are approximate values, the actual volumes delivered depend upon many controllable factors that will vary from one application to another.  For example in liquid to liquid transfers the amount transferred will include the hanging drop on the bottom of the pin as well as the liquid on the sides of the pin.  If the transfer was to a membrane then only the hanging drop would be transferred and not the liquid on the sides of the pin.  If the transfer was to a dry plate then only about half the hanging drop would be transferred to the dry plate and the rest would be on the pin.  The division of the hanging drop would be determined by the surface tension of the pin and the surface tension of the dry plate.

The list on the right summarizes the factors that contribute to the volume delivered.  With each application these factors can be controlled and standardized so that the delivery volumes are very reproducible.  With most applications the CV's are less than 5%.
 
 
1. Pin Diameter
2. Surface tension of the liquid being transferred
3. Surface tension of the pin
4. Speed of removal of pin from source liquid
5. Speed of pin striking recipient dry plate
6. Depth to which the pin is submerged in source plate
7. Depth to which the pin is submerged in recipient plate
8. Volume of slot in pin
9. Surface tension of the dry plate and dwell time
     
 
Q: How can I transfer larger volumes using pins?

A: By placing grooves or slots in the pins that pickup liquids via capillary action. The volume of liquid picked up is directly determined by the size of the groove or slot in the pin.

Our Grooved Pins transfer from 3 ul to 20 ul. They are used to transfer liquid to liquid between a mother plate and a daughter plate.  Both plates must have a liquid level high enough to cover the grooves.

Our Slot Pins transfer from 5 nl to 25 ul. They are used to transfer liquid to liquid between a mother plate and a daughter plate and to make blots on membranes and agar surfaces.  Slot pins also have the advantage of being able to transfer to and from mother and daughter plates that have very low volumes as the slot is in the bottom of the pins and will wick up very tiny volumes in the bottom of the wells if the slot pins are cleaned with the VP 110 Pin Cleaning Solution or have been dipped in ETOH and flamed. 

By increasing the speed with which the pins are removed from the source plate you can increase the volume delivered by as much as 3 fold.

Q: What is the accuracy of delivery using slot pins?

A: In tests transferring 5ml of nitrophenol from one microplate to another using either a 12 channel pipette or a VP 408S5 5ml Slot Pin Replicator, we determined that the accuracy of delivery was much better with the Slot Pin Replicator.  The Coefficient of Variation for the Slot Pin Replicator varied between 2% and 5%.  The 12 channel pipette consistently had a much higher Coefficient of Variation than the Slot Pin Replicator, varying from 8% to 22%.  The mean color intensity and Coefficient of Variation for each row of 12 wells are as follows on the data below.

Row A - pipette 0.312 +/- 16%, .....slot pin 0.301 +/- 5%

Row B - pipette 0.316 +/- 9%,... ....slot pin 0.285 +/- 3%

Row C - pipette 0.337 +/- 16%,......slot pin 0.286 +/- 2%

Row D - pipette 0.315 +/- 8%,........slot pin 0.292 +/- 3%

Row E - pipette 0.303 +/- 22%,......slot pin 0.295 +/- 2%

Row F - pipette 0.358 +/- 16%,......slot pin 0.294 +/- 3%

Row G - pipette 0.399 +/- 11%,......slot pin 0.291 +/- 2%

Row H - pipette (no data)................slot pin 0.296 +/- 2%

More recent testing of the pin to pin precision of slot pins is found in the table below.

 
 


Summary of slot pin to slot pin delivery precision

Pin Diameter Volume of Slot Mother well solvent Recipient well solvent Label CV
1.58 mm 5 ml   Aqueous Aqueous Methyl Orange 3.7%
1.58 mm 2 ml   Aqueous Aqueous Methyl Orange 4.9%
.457 mm 50 hl Aqueous DMSO FITC 3.2%
.457 mm 50 hl ETOH DMSO FITC 3.4%
.457 mm 10 hl Aqueous DMSO FITC 4.0%
.457 mm 10 hl ETOH DMSO FITC 2.7%
.787 mm 100 hl Aqueous Aqueous Horseradish peroxidase 1.6%
.787 mm 200 hl Aqueous Aqueous Horseradish peroxidase 1.9%
 
 


Q: How can I do quantitative studies on high density arrays made on membranes?

A: Although there are several image analysis programs that may be used to determine pixel intensity and total pixel quantitation, we have found that the ImaGeneTM image processing software from BioDiscovery, Inc. provides a very simple and compatible system.  See image of segmented ImaGene display to the right.

 
 
 
Q: Will the stainless steel stirrers react with my reagents?

A: In most cases the stirrers are inert.  However we also provide stainless steel stirrers coated in the polymer Parylene. Parylene is an inert polymer film that makes a conformal coating around the stirrers. Parylene coating is useful in combinatorial chemistry where the reaction may be inhibited by Fe ions from the stirrers.  Coating with parylene further protects the stainless steel stirrers from corrosive effects of a wide variety of concentrated acids and bases.   Parylene is stable from -200C to 200C, it is resistant to organic solvents and it is non-toxic and bio-compatible for medical applications, including implants.   Parylene is very cost efficient and generally only raises the cost of a stirrer about 5 cents. 

We also have stainless steel stirrers coated with PTFE for those special solutions where only Teflon will do.  (TOP)

Q: How should I practice making arrays with Replicators and the registration devices?

A: Just add a dye (food color works fine) to the buffer, media or solutions that you will be arraying.  Do not put the dye into just water as the surface tension of plain water is very high and will give you variable results.  A little DNA, protein or carbohydrate makes a big difference.  If your buffer doesn't or can't contain these substances you can treat the pins with the new V&P Pin Cleaning Solution (VP 110), or dip the pins in ETOH and flame them.   (TOP)

Q: When making arrays with the MULTI-PRINT™ should I use blotting paper under the membrane?

A: Yes.  We sell a wicking paper (VP 521V 22cm x 32cm) that works quite well to support the membrane for 4 MULTI-PRINTS, however Whatman, 3M or other similar paper will also work.  (TOP)

 
         

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