Harris Sampling Tools FAQs
- Where is the best place to take the punch from?
- What size FTA punch do I take?
- Do I need to wash the punch with FTA Purification Reagent?
- How do I process a punch?
- Do I need to dry the punch before PCR?
- Does FTA support qPCR?
- How many punches can I get from an FTA sample circle?
- Why did FTA fail in my PCR?
- Is cross-contamination from the Harris punch a problem?
- How long can a washed punch be stored?
- Is FTA compatible with robotics?
- When using CloneSaver for plasmid work, why doesn't the genomic DNA of the host bacterial cell contaminate the plasmid sample?
- How do I clean the Harris punch?
- How long can I keep and what storage conditions are needed for a washed punch before I use it for PCR/transformation?
- How long can I keep and what storage conditions are needed for eluted plasmid DNA before I use it for PCR/transformation?
- How many punches can I take from a single CloneSaver circle?
- Do I have to worry about cross contamination on CloneSaver?
- What electroporation conditions are recommended?
- I didn't spot into the centre of the circle - the white area is not all in the circle - have I got cross talk and what do I do?
- Can I do restriction digestion of plasmid from CloneSaver (punch or elute)?
- Can I do transfection of plasmid from CloneSaver (punch or elute)?
- Is 2mm the only size punch you can take?
- Does the mat need to be cleaned after each punch?
Where is the best place to take the punch from?
The best place is near the center of the sample area but tests have proven that cells are distributed evenly across the sample area.
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What size FTA punch do I take?
It is recommended to take a 2.0mm punch for buccal cell samples.
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Do I need to wash the punch with FTA Purification Reagent?
Yes.
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How do I process a punch?
Using the normal FTA procedure; wash 3 x 5 min in 200:L FTA Purification then rinse 2 X 5 min in 200:L TE-1 (10mM Tris-HCl, 0.1mM EDTA, pH 8), remove all traces of wash buffer and add PCR master mix to the washed punch.
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Do I need to dry the punch before PCR?
It is not necessary but punches can be dried by incubating at 50oC for 10 min with the tube lid open.
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Does FTA support qPCR?
Quantitative (real-time) PCR does work with immobilized DNA but some thermocycler reaction chambers do not accept the punch. DNA can be eluted for use with these instruments.
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How many punches can I get from an FTA sample circle?
A Classic or Micro Card can give about 140 disks of 1.2 mm diameter or about 50 of 2 mm. A Gene card can give about 60 of 1.2 mm diameter or about 25 of 2 mm diameter.
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Why did FTA fail in my PCR?
Amplification can fail for many reasons so we strongly recommend routine controls. Control punches should be taken from sample-free areas of the FTA card and washed as the sample punches are. One of these no-sample punches is added to a negative control tube. Another goes into a positive control tube with a DNA standard solution. The standard DNA is also used in a positive control tube without any FTA. Results of these controls greatly assist in solving amplification problems.
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Many of these initial problems have been solved by more careful washing to ensure removal of heme from blood samples and chlorophyll and other components from plant samples. Blood and some plant samples also contain a great deal of DNA and so give better results with 1 mm punches than with larger ones. Increasing the PCR reaction volume can also help. Buccal samples sometimes contain primarily saliva rather than cells and so can require larger punches or more cycles of PCR.
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Is cross-contamination from the Harris punch a problem?
No. Dry samples on FTA have not been a problem. This can be confirmed by testing a negative control punch in between two sample punches. If carryover is a particular concern, you can punch an unused area of FTA or blotter paper between samples, or rinse with ethanol and dry with a clean tissue or compressed air.
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How long can a washed punch be stored?
A washed, dried punch can be stored up to one week in the dark at 4 degrees Celsius or below if necessary. This storage is not encouraged since the DNA-protective chemicals have been removed.
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Is FTA compatible with robotics?
Yes. Progress in this area is substantial and ongoing. A Harris e-Core with docking stations is recommended. Customised cards have been and are being developed. Contact your local Whatman sales representatives for more details.
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When using CloneSaver for plasmid work, why doesn't the genomic DNA of the host bacterial cell contaminate the plasmid sample?
Some genomic DNA may be present with the plasmid during analysis, but the gDNA should not contaminate the analysis or affect the results. Both the genomic DNA and the plasmids will bind to the FTA upon sample application. During the washing steps, the cell debris will get washed of, leaving the gDNA and plasmids entrapped within the fibres. If the punch is then used for analysis, the gDNA will be present. If, however, the plasmid is eluted off the punch first, the bacterial genomic DNA will not elute (only eluting in TE for 5 minutes at room temperature) and not be present for analysis.
If the punch is used for PCR or transformation, why wouldn't the gDNA affect analysis? Each of the two common forms of analysis for plasmid, PCR and transformation, are specific to plasmids. The primers used during PCR should be specific for regions on the plasmid and not for the genomic DNA. Transformation involves inserting DNA into awaiting host cells that have been made 'sick' - fragile to the point of having the cell walls disrupted. The genomic DNA will not be available for transformation as the molecule will be tightly entrapped within the fibres of the FTA punch. Those molecules of gDNA that happen to get transformed will not survive long in the new cell as they are not able to self-replicate like plasmids do. For these reasons, any contaminating gDNA will have no impact on either PCR or transformation.
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How do I clean the Harris punch?
It can be cleaned in three different ways. Use whichever you find works best in your lab: Punch into an unused portion of the CloneSaver Card or clean filter paper between sample Wash the tip with alcohol between samples Spray the tip with canned/compressed air between samples and wipe with a clean KimWipe.
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How long can I keep and what storage conditions are needed for a washed punch before I use it for PCR/transformation?
The washed punch can be stored for one week at 4 degrees Celsius and still be used for PCR analysis. However, perform transformations as soon as possible after washing.
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How long can I keep and what storage conditions are needed for eluted plasmid DNA before I use it for PCR/transformation?
DNA eluted off of the punch can be stored for one week at 4 degrees Celsius and still be used for PCR analysis. However, perform transformations as soon as possible after elution.
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How many punches can I take from a single CloneSaver circle?
If taken from the inside edges of the circle, four 2.0 mm punches or at least seven 1.2 mm punches can be obtained from one circle on the card.
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Do I have to worry about cross contamination on CloneSaver?
As long as samples were spotted within the printed circles and the punches are taken from within the printed circles, there should be no cross contamination of samples.
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What electroporation conditions are recommended?
For electroporation, we recommend the standard conditions specified for the electrocompetent cells and for the electroporation apparatus. There is no need to change the conditions for transformations using either the CloneSaver punch or the DNA eluted from the punch.
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I didn't spot into the centre of the circle - the white area is not all in the circle - have I got cross talk and what do I do?
Punch into a white portion within the circle from which you want to recover the clone. There is not cross-contamination between circles if spotted from within the line. The ink of the circle does not interfere with downstream applications.
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Can I do restriction digestion of plasmid from CloneSaver (punch or elute)?
There is not enough DNA from a punch or eluted off of the punch to perform restriction digestion, and it will be contaminated with genomic DNA from the bacteria.
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Can I do transfection of plasmid from CloneSaver (punch or elute)?
This cannot be done from either a punch or the DNA eluted off of the punch from CloneSaver. The system does not remove endotoxin.
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Is 2mm the only size punch you can take?
No. The 2.0 mm punch provides higher yield of colonies than the 1.2 mm punch, and the 2.0 mm punch has been used in all of the validation studies. However, adequate retrieval of clones has been achieved with the 1.2 mm punch.
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Does the mat need to be cleaned after each punch?
The mat does not need to be cleaned in between punches as long as the CloneSaver Card is not moved around too much on the mat while sampling. However, you should clean the mat with alcohol in between cards to prevent cross-contamination.
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