Fta Dna Extraction

FTA Protocols:
Collect, Transport, Archive

And Access Nucleic Acids
All at Room Temperature
Whatman
For research use only. Not for diagnostic

procedures.
WB120047
06/02
Whatman 2002
Table of Contents:
1.
2.
3.
4.
5.
For research use only. Not for diagnostic

procedures.
Introduction: FTA technology1

Handling guidelines... 2
General protocol overview 3
Removing a sample disc from a card for analysis ... 4
Applying samples to FTA Cards and preparation for PCR
a. Blood samples. 5
b. Buccal cell samples.8
c. Tissue/cell culture samples..11
d. Plant samples.13
e. Bacterial cultures.. 15
f. Mouse tails..17
6. Preparation for downstream applications
a. RFLP..20
b. STR.23
c. RT-PCR/Northern Blot. 25
7. CloneSaver Card
a. Introduction.27
b. Application of sample28
c. Storage of samples28
d. Preparation of sample for transformation or PCR 29
e. Transformation (using a punch). 29
f. Transformation (using DNA eluted from punch)30
g. PCR from a CloneSaver punch.. 31
8. Related publications. 33
9. Ordering information. 35
10. Contact information.. 36
1. Introduction: FTA Technology
FTA technology is a novel method designed to simplify the

collection, shipment, archiving and purification of nucleic acids
from a wide variety of biological sources. These include (but are
not limited to) whole blood, buccal cells, plant material, tissue
cells/culture, microorganisms, and plasmids.
FTA Cards are impregnated with a patented chemical formula that
lyses cell membranes and denatures proteins upon contact.
Nucleic acids are physically entrapped, immobilized, and
stabilized for storage at room temperature. FTA Cards protect
nucleic acids from nucleases, oxidation, UV damage and
microbial and fungal attack. Infectious pathogens in samples
applied to FTA Cards are rendered inactive upon contact.
Samples collected on FTA Cards can be stored at room
temperature; the nucleic acids are stable for years. The stability
of genomic DNA on FTA Cards for at least 11 years has been
demonstrated.
2. FTA Handling Instructions:
Always wear gloves when handling FTA Cards or

CloneSaver Cards to avoid contamination of the cards.
Store unused FTA/CloneSaver Cards in a cool, dry place
(avoid light and excessive humidity).
Follow universal precautions when working with biological
samples.
FTA Cards and CloneSaver Cards are non-toxic to
humans.
Samples stored on FTA Cards and enclosed in a multi-barrier

pouch can be shipped through the US Postal Service with no
special handling restrictions, making them an extremely useful
tool for field collection of blood, plants or other specimens.
Indicating FTA Cards turn from pink to white upon sample
application and are recommended for clear or colorless samples.
CloneSaver Cards are optimized for the room temperature
collection and storage of plasmid DNA.
1
3. General Protocol for PCR Preparation
4. Removing a sample disc from an FTA or

CloneSaver Card for preparation and analysis of
DNA
Apply sample to the FTA Card.
Remove disc (typically

1.2 mm or 2.0 mm).
See page 4.
Place disc in reaction
tube. Wash disc with
FTA Purification Reagent.
Wash disc with TE-1 Buffer.
Air-dry the disc.
Add PCR Master Mix

directly to the disc
and proceed to PCR.
3
1. Always ensure that the sample applied (see pages 5-19) is

dry before taking a punch.
2. Place the FTA Card/CloneSaver Card on a cutting mat. For
cards with outer paper layers, ensure the mat is directly
beneath the FTA Card with no paper layer in between.
3. Place the tip of a coring device, e.g., a Harris 1.2 mm or
2.0 mm Micro-Punch, over the area to be sampled. Do NOT
depress the ejection plunger at this time.
4. Press down firmly on barrel of the coring device and twist to
cut a disc out of the card.
5. Once the disc is in the corer, transfer the disc to the desired
PCR tube or tray by depressing the ejection plunger and
ejecting the disc.
6. In order to ensure there is no cross-contamination between
samples, the coring device can be cleaned using three
methods. Use the method that best fits your laboratory
workflow:
Cleaning the corer tip:
a. Rinse the tip with ethanol between samples and dry with
a sterile wipe, or
b. Take one punch from blank filter paper or an unspotted
area of the FTA/CloneSaver card between samples, or
c. Clean the tip of the punch with a blow of compressed air
between samples.
4
5.a. Applying and Preparing Blood Samples on

FTA Cards: (Also for applying bone marrow
aspirate, buffy coats and plasma samples)
Blood samples from fingerstick whole blood, and blood collected
with the following anticoagulants: EDTA, sodium citrate, ACD, or
heparin can be stored on FTA.
Sample Application to FTA Cards:
1. Label the FTA card with the appropriate sample identification.
2. Drop the blood (<125 l per 1 inch circle; 75 l per GeneCard
circle) onto the card in a concentric circular motion within the
spotted circle. Avoid puddling of the liquid sample, as it will
overload the chemicals on the card. Also, do not rub or
smear the blood onto the card.
3. Allow the samples to dry for about one hour at room
temperature. Do NOT heat assist the sample drying step, as
this may fix PCR inhibitors onto the matrix.
4. Dried blood spots will appear much darker than freshly
spotted ones.
5. If the sample is to be archived, place card in a Multi-Barrier
pouch with desiccant or store in a humidity controlled, cool,
dry environment.
Preparation for PCR analysis: refer to page 6.
Preparation for RFLP analysis: refer to page 20.
Preparation for STR analysis: refer to page 23.
5
Preparing an FTA Disc for PCR Analysis:

1. Take a sample disc from the dried spot following the
instructions on page 4. Place disc in PCR amplification tube.
For blood samples, a 1.2 mm disc in a 25 l PCR reaction is
recommended.
2. Add 200 l of FTA Purification Reagent to PCR tube.
3. Incubate for 5 minutes at room temperature. (The tube may
be given moderate manual mixing if desired.)
4. Remove and discard all spent FTA Purification Reagent using
a pipette.
5. Repeat steps 2-4 twice, for a total of three washes with FTA
Purification Reagent.
6. Add 200 l of TE-1 Buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH
8.0).
7. Incubate for 5 minutes at room temperature.
8. Remove and discard all spent TE-1 Buffer with a pipette.
9. Repeat steps 6-8 once for a total of two washes with TE-1
Buffer.
10. Allow disc to dry at room temperature for about one hour (or
at 56C for 10 minutes) before performing PCR.
PCR Analysis:
The washed and dry disc is now ready for PCR analysis using
standard protocols. The disc is included in the PCR reaction.
There is no need to change reaction volume or PCR conditions
due to the presence of the disc.
Alternative Method: Fast Centrifugation Preparation Protocol

for PCR Analysis:
1. Take a sample disc from the desired blood spot following the
instructions on page 4. Place disc in a spin basket of a
microcentrifuge tube. For blood samples, a 1.2 mm disc in a
25 l PCR reaction is recommended.
2. Add 200l FTA Purification Reagent to each spin basket,
cap, and incubate 1 minute at room temperature.
3. Centrifuge tubes at 12,000 x g for 10 seconds.
4. Repeat steps 2 and 3 for a total of two FTA Purification
Reagent washes.
5. Add 200l of DNAase free water to each spin basket, cap,
and incubate for 1 minute at room temperature.
6. Centrifuge tubes at 12,000 x g for 10 seconds.
7. Remove the disc from the spin basket, it is now ready for
PCR analysis.
5.b. Applying and Preparing Buccal Cell Samples

on FTA Cards:
To capture the optimum amount of DNA on the FTA Card, the
following collection and transfer of buccal cells to FTA Cards is
recommended. Indicating FTA Cards are recommended for use
with buccal cell samples.
Sample Collection and Application to FTA Cards:
1. Place the Indicating FTA Card on a clean, dry, flat surface.
Label the FTA Card with a unique identifying name or
number.
2. Remove one Foam Tipped Applicator from the protective
packaging according to instructions on the packaging.
3. Holding the plastic handle of the Applicator, place the foam tip
in the mouth and rub one side of the foam tip on the inside of
the cheek for 30 seconds. Repeat using the opposite side of
the foam tip for the other cheek. Run the foam tip along the
gum-line and fold of the cheek and under the tongue, soaking
up as much saliva as possible. Remove the Applicator from
the mouth.
4. Carefully lift the paper cover of the Indicating FTA Card to
expose the pink sample area. Press the flat, circular foam
Applicator tip within the sample circle area. Without lifting the
foam tip from the card, squeeze the tip using a side-to-side
motion (90 in each direction) 3 times to completely saturate
the sample area. Turn the Applicator over and repeat with
the other side of the foam tip within the same circle. The
sample area will turn white upon transfer of the sample.
8
5. Discard the Applicator according to laboratory procedure. Do

not place the foam swab into the mouth after it has touched
the FTA Card.
6. Position the FTA Card for drying by supporting the sample
area with the paper cover as shown.
Allow the card to dry
for at least 1 hr at room temperature.
7. If the sample is to be archived, place in
a Multi-Barrier pouch with desiccant or store
in humidity controlled, cool, dry environment.
If buccal cells are to be applied to more than one FTA sample
area, use a new Applicator and repeat steps 1 - 6.
Preparation for PCR: refer to page 10.
Preparation for STR analysis: refer to page 23.

1. Take a sample disc from the dried spot on the Indicating FTA
Card following the instructions on page 4. Place disc in PCR
amplification tube. For buccal cell samples, a 2.0 mm disc is
recommended.
3. Incubate for 5 minutes at room temperature, with moderate
manual mixing if desired.
a pipette.
8.0).
Buffer.
PCR Analysis:
There is no need to change reaction volume or PCR
conditions due to the presence of the disc.
10
5.c. Applying and Preparing Tissue / Cell Culture

Samples on FTA Cards:
Sample Application to FTA Card:
1. Tissue culture cells should be applied to FTA Cards in a
minimum concentration of 100 cells/l in media, trypsin, or
PBS buffer. About 65 l of sample will fill one circle on an
FTA card.
2. Allow the samples to dry for about one hour at room
temperature. Do NOT heat assist the sample drying step, as
this may fix the PCR inhibitors to the matrix.
3. If the sample is to be archived, place in a Multi-Barrier pouch
with desiccant or store in a humidity controlled, cool, dry
environment.
A 2.0 mm disc is recommended for tissue or cell culture
samples with low cell counts (~100 cells/l). For high cell
concentration (~1,000 cells/l), a 1.2 mm disc is
recommended.
a pipette.
11

8.0).
Buffer.
10. Allow disc to dry at room temperature for about one hour, (or
PCR Analysis:
Related sample types:
Tumor cells and solid tissues such as liver have been applied to
FTA Cards with successful PCR amplifications.
For additional information, the following publications may be useful:
Dobbs et al, 2002. Use of FTA Gene Guard Filter Paper for the
Storage and Transportation of Tumor Cells for Molecular Testing.
Archives of Pathology and Laboratory Medicine 126:56-63.
Higgins et al, 2000. Detection of Francisella tularensis in Infected
Mammals and Vectors Using a Probe Based Polymerase Chain
Reaction. Am. J. of Tropical Medicine and Hygiene 62:310-318.
12
5.d. Applying and Preparing Plant Tissues on FTA

Cards:
Direct Leaf Press:
1. Place leaf material directly onto the FTA card, and cover the
leaf with parafilm.
2. Apply moderate pounding/pressure with a blunt object such
as a tack hammer or pestle.
3. The collection process is considered complete when the
extract is drawn through to the back of the FTA card.
4. Allow plant samples on FTA to dry for at least an hour at
room temperature. Do NOT heat-assist the drying period.
5. If sample is to be archived, place in a Multi-Barrier pouch with
desiccant or store in a humidity controlled, cool, dry
environment.
Plant Homogenate:
1. Use about 10-20 mg of plant tissue for the homogenate.
2. Add PBS buffer to plant tissue using an estimated ratio of 1
part plant tissue to 5 parts PBS buffer. Grind until it is
apparent that some plant tissue is homogenized. The
homogenate does not have to have a smooth consistency.
3. Using a pipette, apply about 25 l of plant homogenate to
each circle on an FTA card.
4. Allow plant homogenate on FTA to dry for at least an hour at
room temperature. Do NOT heat-assist the drying period.
environment.
13
Preparing an FTA Disc for PCR Analysis (for leaf press or

homogenate):
A 2.0 mm disc is recommended for plant samples.
a pipette.
5. Repeat steps 2-4 once, for a total of two washes with FTA
8.0).
Buffer.
PCR Analysis:
14
5.e. Applying or Preparing Bacteria on FTA Cards:

A 1.2 mm disc is recommended for use with genomic DNA
from bacteria. For plasmid DNA analysis, a 2.0 mm disc is
recommended.
a pipette.
5. Repeat steps 2-4 once for a total of two washes with FTA
8.0).
Buffer.
10. Allow punch to dry at room temperature for about one hour,
(or at 56C for 10 minutes) before performing PCR.
Bacterial Colonies
1. Pick a single colony from agar, and resuspend in 5-10 l of
bacterial culture medium, PBS, or TE-1 buffer.
2. Apply 5-10 l of bacterial suspension to FTA (Indicating FTA
Cards are recommended). If applied to non-Indicating FTA
cards, circle the application spot with a ballpoint pen or pencil.
3. Allow sample to dry for at least one hour at room
temperature.
environment.
Overnight Bacterial Cultures:
1. Grow bacteria overnight in medium.
2. Apply approximately 65 l of culture to FTA (Indicating FTA
Card is recommended). If applied to non-Indicating FTA,
circle the application spot with a ballpoint pen or pencil.
3. Allow sample to dry for at least one hour at room
temperature.
environment.
PCR Analysis: The washed and dry disc is now ready for PCR
analysis using standard protocols. The disc is included in the
PCR reaction. There is no need to change reaction volume or
PCR conditions due to the presence of the disc.
15
For information on PCR ribotyping of bacteria, refer to:

Rogers and Burgoyne, 1997. Bacterial Typing: Storing and Processing of
Stabilized Reference Bacteria for PCR Without Preparing DNA- An example of
an Automatable Procedure. Analytical Biochemistry 247:223-227.
16
5.f. Applying and Preparing Mouse Tails on FTA

Cards:
5. Avoiding any pieces of tissue, apply 5 l of digested tail

solution to FTA card (Indicating FTA is recommended). If
not applied to Indicating FTA, circle the application spot
with a ballpoint pen or pencil.
6. Allow the sample to dry for at least an hour at room
temperature. Do not heat assist drying.
7. If the sample is to be archived, place in a Multi-Barrier
pouch with desiccant or store in a humidity controlled,
cool, dry environment.
Mouse Tail Blood:

1. Snip approximately 1.5 cm of the mouse tail.
2. The tail blood can either be immediately applied to the FTA
card, or the tail can be placed in a sterile microcentrifuge tube
and transferred on ice to the laboratory. The blood should be
placed on FTA within 2 hours of collection.
3. Gently squeeze the tail clipping to bring blood to the cut
surface, and dab the cut surface to the FTA card to transfer
the blood. About 3-4 drops of blood can be obtained per tail.
If no blood is present, please see the Mouse tails, Digested
instructions below.
environment.
Mouse Tails, Digested:
1. Obtain fresh mouse tails, or tails that have been
preserved in ethanol and stored at 20C.
2. Using clean forceps, hold mouse tail and drain excess
ethanol.
3. Transfer the tail section into a 1.5 mL microcentrifuge
tube and allow it to air dry until ethanol evaporates.
4. Add 600 l of TNES Buffer (10 mM Tris, pH 7.5, 400 mM
NaCl, 100 mM EDTA, 0.6% SDS) and 35 l of Proteinase
K (10 mg/ml) to the tube and incubate for 8-24 hours at
55C.
Preparation for PCR analysis: refer to page 19.

Preparation for RFLP analysis: refer to page 20.
17
18
Sample Preparation for PCR Analysis:
1. Take a sample disc from the desired blood or tissue digest
spot following the instructions on page 4. Place disc in PCR
amplification tube. For mouse tail samples, a 1.2 mm disc in a
25 l PCR reaction is recommended.
a pipette.
8.0).
Buffer.
10. Allow disc to dry at room temperature for about an hour, (or at
56C for 10 minutes) before performing PCR.
PCR Analysis:
19
6.a. Purification Protocol for Downstream RFLP

Analysis of Sample DNA on an FTA Card:
Materials Required:
FTA Purification Reagent
2.0 ml microcentrifuge tube with spin basket insert
Microcentrifuge capable of speeds up to 12,000 x g
Proteinase K (10 mg/mL) or DNAzol Reagent (Invitrogen)
TE-1 Buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0)
Restriction endonuclease stock Hae III (10 U/l)
10X restriction digest buffer
Sterile deionized water
6 mm or 7 mm diameter paper puncher
37c incubator; 60c incubator
Protocol:
1. Remove a punch (6 mm or 7 mm) from a dried FTA Card and
place it in the bottom of the basket insert. Make sure the
punch is firmly in contact with the basket
2. Place the basket into the 2.0 mL-microcentrifuge tube
3. Add 500 l FTA Purification Reagent to the basket. Make
sure the disc is on the bottom of the basket.
4. Incubate for 1 minute at room temperature
5. Centrifuge at 6,000 x g for 30 seconds.
6. Remove the basket and decant spent Reagent.
20
7. Return the basket to the microcentrifuge tube and repeat
Steps 3-5 twice for a total of three washes with the FTA
8. Add 495 L FTA Purification Reagent and 5 l
Proteinase K (10 mg/mL) to each tube.
Note: 500 l of DNAzol Buffer can be substituted for the
above buffer combination.
21
18. Prepare a 1X restriction digest master mix for a final
volume of 50 l:
For every 50 L desired combine:
5 L of 10X restriction digestion buffer
50 units of restriction enzyme
(typically 5 l of a 10 U/l stock)
Bring volume up to 50 l by adding sterile, ddH2O.
9. Incubate for one hour at 60C.

Note: if using DNAzol Buffer, instead of Proteinase K,
incubate for 5 minutes at room temperature.
19. Place basket in a clean 2 mL tube
10. Centrifuge at 6,000 x g for 30 seconds.
22. Centrifuge at 12,000 x g for two minutes.
11. Remove the basket and decant spent reagent/buffer.

12. Return the basket to the microcentrifuge tube.
13. Add 500 l of TE-1 Buffer to the tube.
14. Centrifuge at 6,000 x g for 30 seconds
15. Remove the basket and decant spent buffer.
16. Return the basket to the microcentrifuge tube
17. Repeat steps 12-15 twice for a total of three washes with
TE-1 Buffer.
20. Add 50 l of master mix to each basket

21. Incubate for two hours at 37C.
Note: The restricted DNA is recovered in the microcentrifuge tube.
23. Collected DNA can now be analyzed by desired RFLP

methods.
23
22
6.b. Preparation of samples from FTA Cards for

STR (Short Tandem Repeats) Analysis:
1. Take a sample disc from the desired blood spot following
the instructions on page 4. Place disc in appropriate
reaction tube. A 1.2 mm disc is recommended.
3. Incubate for 5 minutes at room temperature, with
moderate manual mixing if desired.
4. Remove and discard all spent FTA Purification Reagent
using a pipette.
5. Repeat steps 2-4 twice, for a total of three washes with
FTA Purification Reagent.
6. Add 200 l of TE-1 Buffer (10 mM Tris-HCl, 0.1 mM
EDTA, pH 8.0).
Buffer.
10. Allow disc to dry at room temperature overnight prior to
analysis.
STR Notes:
Applied Biosystems (ABI) AmpFSTR Identifiler Kit

recommends only one wash with FTA Purification Reagent
and 2 washes with TE-1 Buffer. A 1.2 mm disc contains about
5-20 ng DNA. Accordingly, an appropriate cycle number for
this high quantity of DNA is 25 cycles, amplified directly in the
MicroAmp tube.
Other STR profiles have been successfully achieved with 1.2
mm FTA discs using the GenePrint PowerPlex 1.1 and 2.1,
and the PowerPlex 16 BIO System.
24
6.c. Preparation of RNA from Blood and Tissue

Culture on FTA Cards for RT-PCR or Northern Blot
Analysis
Unlike genomic DNA stored on FTA cards, RNA can be eluted
from the FTA matrix. The RNA that elutes from the FTA matrix is
suitable for RT-PCR and Northern Blot Analysis. As little as 1/80th
of the eluate can be used for the first strand synthesis reaction.
(Trace amounts of genomic DNA may also elute from this punch,
so it is important to have a negative control if this sample will be
used for RT-PCR).
Note: To isolate sufficient RNA for Northern Blot Analysis,
(approximately 300 ng) the entire spot of cells (50 l or 4.25 x 107
cells/ml) needs to be processed. To account for this larger
sample size, increase wash step to 750 l of sterile water.
Materials Required:
RNA Processing buffer: (10 mM Tris-HCl, pH 8.0, 0.1 mM
EDTA, 800 U/mL RNase Out(Invitrogen) 200 g/mL
glycogen, and 2 mM DTT). Always make this solution fresh
and store on ice.
Ice Bath
7.5 M ammonium acetate or 3 M sodium acetate pH 5.2
100% isopropanol, molecular biology grade
75% ethanol, molecular biology grade
Microcentrifuge
25
Protocol:
2. Add 400 l of RNA Processing Buffer to the tube and pipette
up and down twice, cap, and incubate for 15 minutes on ice,
mixing every 5 minutes. The RNA is eluted from the FTA disc.
3. Precipitate the RNA from the RNA processing buffer using
method A or B.
Method A: Add volume of 7.5 M ammonium acetate, mix,
then add an equal volume of ice cold 100% isopropanol.
Method B: Add 1/10th volume of 3 M sodium acetate, pH 5.2,
mix, then add an equal volume of ice cold 100% isopropanol.
4. Incubate at 20C for 1 hour.
5. Place tube in microcentrifuge tube and spin down at
12,000 x g. Remove supernatant.
6. Wash pellet with 500 l of ice-cold 75% ethanol and spin for
5 minutes at 12,000 x g.
7. Remove supernatant and air dry the pellet.
8. Resuspend the pellet in 50 l of TE-1 buffer. Now the isolated
RNA is suitable for RT-PCR or Northern Blot Analysis.
9. Follow standard procedures for RT-PCR or Northern Blot
analysis.
26
7.a. Storing and Retrieving Plasmid DNA from

CloneSaver Cards:
Introduction
The CloneSaver Card is designed for the long-term, room
temperature storage of plasmid and BAC DNA clones. Purified
plasmid DNA can also be stored on the CloneSaver. DNA stored
on the CloneSaver Card is available for bacterial transformations
and PCR amplifications. The sample area is formatted in a 96well configuration. Samples applied to the Card as directed will
not contaminate other samples in adjacent circles. The pink color
of the CloneSaver changes to while upon application of liquid
samples allowing easy identification of sample location.
Samples that can be applied to CloneSaver include overnight
cultures of bacteria, suspended colonies, glycerol stocks and
purified plasmid DNA.
The CloneSaver Card protects clones from bacteriophage
contamination and allows rescue of bacteriophage infected
cultures.
Please follow the same handling/storage guidelines as FTA
products, found on page 2.
27
7.b. Application of Bacterial Samples:

1. Unfold the CloneSaver Card to expose the pink matrix card
(96 well format).
2. Pipette 5 l of an overnight bacterial culture, e.g., OD600 ~1.8
in LB, into the center of one of the 96 printed circles.
Bacterial colonies can be applied by first resuspending a
single colony in 5 l of sterile growth media or sterile TE-1
buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) and then spotting
the sample to the card. The pink color of the matrix will turn
white upon sample application. To prevent cross
contamination, apply no more than 5 l of sample to each
circle. (Bacterial glycerol stocks and purified plasmid DNA
can also be applied to CloneSaver Cards. Call Technical
Service for details.)
3. After applying sample, label the corresponding area of the
CloneSaver ID Chart.
4. Let samples air-dry at least 1 hour prior to storage or
processing. Heat-assisted drying is not recommended. Avoid
contact with potential contaminants while drying.
5. Once completely dry, fold the CloneSaver Card so that the
pink matrix card is turned down facing the Quick Directions.
The cover can be closed over the turned down card.
7. c. Storage of Plasmid or BAC DNA on CloneSaver Cards:
Plasmid or BAC DNA on CloneSaver Cards should be stored at
room temperature in a cool, dry place (avoid light and excessive
humidity). The recommended storage procedure is to place the
CloneSaver Card in a Resealable Multi-Barrier Pouch with a
Storage Desiccant .
28
7.d. Preparation of CloneSaver Punch for Transformation or

PCR:
1. Remove a 2.0 mm punch from the target sample using a
Harris Micro-Punch or a similar device according to its
instructions. It is important to ensure that no residual material
is carried from one punch to the next. See Cleaning the
corer tip, page 4.
2. Transfer the CloneSaver punch to a tube suitable for
transformation or PCR.
3. Wash the punch by adding 200 l of sterile TE-1 buffer to the
tube and gently pipetting the buffer up and down twice.
Completely remove and discard the TE-1 buffer.
Note: To minimize plasmid loss, do not let the punch remain
in TE-1 buffer longer than necessary for the washing step.
4. Repeat step 3 and then remove all traces of TE-1 buffer.
The washed punch can now be used for transformation or
PCR as described below. If the CloneSaver punch needs to
be transferred to a different tube, use a sterile pipette tip.
7.e. Transformation Using a Washed CloneSaver Punch:
For plasmid DNA, a washed CloneSaver punch can be used to
transform either electrocompetent cells or chemically competent
cells. For BAC DNA, transformation using a punch with
electrocompetent cells is recommended.
Note: We recommend that transformation by electroporation to be
performed using the elution method, unless it is a low copy
number plasmid. Please see 7.f. Transformation Using Plasmid
DNA Eluted from a CloneSaver Punch, page 30.
29
1. For the heat shock method of transformation, carefully pipette

chemically competent cells directly to the tube containing the
washed CloneSaver punch. Perform the transformation
according to standard protocols.
2. For extremely low copy number plasmids use transformation
by electroporation directly off the punch. Place the tube
containing the washed CloneSaver punch on ice. Add
electrocompetent cells directly to the tube and incubate, on
ice, for 10 minutes. Transfer the cells and punch to a chilled
electroporation cuvette. Perform transformation according to
standard electroporation protocols.
Note: Electroporation can also be performed using DNA
eluted from the punch as described below.
7.f. Transformation Using Plasmid DNA Eluted from a
CloneSaver Punch:
Sufficient plasmid DNA can be eluted off the CloneSaver punch
for transformation by electroporation. This procedure should not
be used with BACs stored on CloneSaver Cards or for
transformation using chemically competent cells.
Preparation of Eluted DNA from a CloneSaver Punch:
1. Prepare a washed punch as described in Preparation of
CloneSaver Punch for Transformation or PCR, Steps 1-4.
2. Add 5 l sterile TE-1 buffer to the punch in the wash tube.
Incubate for 10 minutes at room temperature.
Transformation Using Eluted DNA
1. Transfer 2 l of the eluted DNA to a tube containing
electrocompetent cells.
2. Perform the transformation according to standard
electroporation protocols.
30
NOTE: Transformation efficiency will vary with copy number,

plasmid size, concentration of cells spotted, and efficiency of the
competent cells used. Transformation of high efficiency
competent cells (with high copy number plasmids up to 10 kb in
size) will produce up to 10,000 colonies when 100 l of a 1ml
transformation is spread on selective agar plates.
7.g. PCR Using Either a Washed CloneSaver Punch or DNA
Eluted from a CloneSaver Punch:
Sample Preparation
Washed Punch: Prepare a washed punch as described in
Preparation of CloneSaver Punch for Transformation or
PCR, steps 1-4. Add PCR reaction mixture (25 l) directly to
the punch.
Eluted DNA: Prepare eluted DNA as described in
Preparation of Eluted DNA from a CloneSaver Punch. When
DNA is eluted from a CloneSaver Card as described, 1 l of
eluted DNA can be used in a 25 l PCR reaction.
PCR Procedure
Follow standard PCR protocols. No alterations in the reaction mix
or cycling conditions are required.
31
32
8. Related Publications:
Rogers et al, 1998. Implementation of a Buccal Swab

Kit for the Collection of DNA Database Samples.
Poster: 9th Annual Int. Symp. On Human Identification
Orlando, FL, USA
Zhong et al, 2001. Comparison of IsoCode STX and

FTA Gene Guard Collection Matrices as Whole Blood
Storage and Processing Devices for the Diagnosis of
Malaria by PCR. Journal of Clinical Microbiology
39:1195-1196
For FTA Products:

Beck et al, 2001. Simple, Sensitive, and Specific
Detection of Human Immunodeficiency Virus Type 1

Subtype B DNA in Dried Blood Samples for the
Diagnosis in Infants in the Field. Journal Clinical
Microbiology 39: 29-33
Belgrader et al, 1995. Automated DNA Purification

and Amplification from Bloodstain Cards Using a
Robotic Workstation. BioTechniques 19: 427-432
Del Rio et al, 1996. Reusing the Same Blood-Stained

Punch for Sequential DNA Amplifications and Typing.
BioTechniques 20: 970-974
Karle et al, 2001. Novel filter-based technology for

simple collection and preparation of PCR-ready plant
DNA: applications for transgenic plant detection.
Poster: Plant and Animal Genome Conference, San
Diego, USA
Orlandi & Lampel, 2000. Extraction Free, Filter-based

Template Preparation for Rapid and Sensitive PCR
Detection of Pathogenic Parasitic Protozoa. Journal
Clinical Microbiology 38: 2271-2277
33
For CloneSaver Card:
Igoe et al, 2001. A Novel Automatable System for the

Room Temperature Archiving and Recovery of DNA
Clones. Poster: Genome Sequencing and Analysis
Conference, San Diego, USA
Goldsborough et al, 2000. Room Temperature

Archiving of Plasmid Clones in an Automatable 96Well Format. Poster Abstract: Plant and Animal
Genome VIII San Diego, USA
34
9. Ordering information
10. Contact Information
Whatman Catalog Numbers:
United States
Product
FTA Classic Card
Indicating FTA Classic Card*
FTA Mini Card
Indicating FTA Mini Card*
FTA Micro Card
Indicating FTA Micro Card*
FTA Gene Card
FTA Purification Reagent
Harris Micro Punch 1.2mm
(with Mat)
Harris Micro Punch 1.2mm Tip
Harris Micro Punch 2.0mm
(with Mat)
Harris Micro Punch 2.0mm Tip
Cutting Mat
Multi-Barrier Pouch (large) 9
x 15 cm
Multi-Barrier Pouch (small) 8
x 7 cm
Flat FTA Card Mailer
CloneSaver Card
CloneSaver Resealable MultiBarrier Pouch
Desiccant
Quantity
100/pack
100/pack
100/pack
100/pack
100/pack
100/pack
100/pack
500 mL
Catalog number
WB12-0205
WB12-0206
WB12-0055
WB12-0056
WB12-0210
WB12-0211
WB12-0208
WB12-0204
1
1
WB10-0005
WB10-0006
1
1
1
WB10-0007
WB10-0008
WB10-0020
500/pack
WB10-0010
500/pack
50
5/pack
WB10-0011
WB10-0016
WB12-0028
50/pack
1000/pack
WB10-0024
WB10-0003
For Forensics / Human ID Applications:

Tel: 1-866-PureDNA
Fax: 1-877-625-1020
Email: bioscience@whatman.com
For Research Applications:

Tel: 1-800-631-7290
Email: info@whatman.com
Europe
35
Japan
Tel: +44 (0) 1622 676670

Fax: +44 (0) 1622 677011
information@whatman.co.uk
Tel: +81 3 3832 6707

Fax: +81 3 3832 6457
japaninfo@whatman.co.jp
Asia Pacific
Rest of World
Tel: +65 6534 0138

Fax: +65 6534 2166
wap@whatman.com.sg
*Indicating FTA Cards turn from pink to white upon sample

application and are recommended for clear or colorless samples.
Fax: 1-973-773-0168
information@whatman.co.uk
Whatman
www.whatman.com
36

Fta Dna Extraction

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Fta Dna Extraction

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FTA Protocols:

Collect, Transport, Archive

For research use only. Not for diagnostic

For research use only. Not for diagnostic

Introduction: FTA technology1

1. Introduction: FTA Technology

FTA technology is a novel method designed to simplify the

2. FTA Handling Instructions:

Always wear gloves when handling FTA Cards or

Samples stored on FTA Cards and enclosed in a multi-barrier

3. General Protocol for PCR Preparation

4. Removing a sample disc from an FTA or

Apply sample to the FTA Card.

Remove disc (typically

Wash disc with TE-1 Buffer.

Air-dry the disc.

Add PCR Master Mix

1. Always ensure that the sample applied (see pages 5-19) is

5.a. Applying and Preparing Blood Samples on

Preparing an FTA Disc for PCR Analysis:

Alternative Method: Fast Centrifugation Preparation Protocol

5.b. Applying and Preparing Buccal Cell Samples

5. Discard the Applicator according to laboratory procedure. Do

Preparing an FTA Disc for PCR Analysis:

5.c. Applying and Preparing Tissue / Cell Culture

6. Add 200 l of TE-1 Buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH

5.d. Applying and Preparing Plant Tissues on FTA

Preparing an FTA Disc for PCR Analysis (for leaf press or

5.e. Applying or Preparing Bacteria on FTA Cards:

Preparing an FTA Disc for PCR Analysis:

For information on PCR ribotyping of bacteria, refer to:

5.f. Applying and Preparing Mouse Tails on FTA

5. Avoiding any pieces of tissue, apply 5 l of digested tail

Mouse Tail Blood:

Preparation for PCR analysis: refer to page 19.

6.a. Purification Protocol for Downstream RFLP

9. Incubate for one hour at 60C.

19. Place basket in a clean 2 mL tube

10. Centrifuge at 6,000 x g for 30 seconds.

22. Centrifuge at 12,000 x g for two minutes.

11. Remove the basket and decant spent reagent/buffer.

20. Add 50 l of master mix to each basket

23. Collected DNA can now be analyzed by desired RFLP

6.b. Preparation of samples from FTA Cards for

Applied Biosystems (ABI) AmpFSTR Identifiler Kit

6.c. Preparation of RNA from Blood and Tissue

7.a. Storing and Retrieving Plasmid DNA from

7.b. Application of Bacterial Samples:

7.d. Preparation of CloneSaver Punch for Transformation or

1. For the heat shock method of transformation, carefully pipette

NOTE: Transformation efficiency will vary with copy number,

Rogers et al, 1998. Implementation of a Buccal Swab

Zhong et al, 2001. Comparison of IsoCode STX and

For FTA Products:

Detection of Human Immunodeficiency Virus Type 1

Belgrader et al, 1995. Automated DNA Purification

Del Rio et al, 1996. Reusing the Same Blood-Stained

Karle et al, 2001. Novel filter-based technology for

Orlandi & Lampel, 2000. Extraction Free, Filter-based

For CloneSaver Card:

Igoe et al, 2001. A Novel Automatable System for the

Goldsborough et al, 2000. Room Temperature

10. Contact Information

Whatman Catalog Numbers: