Overview

Bacteriophage T4 was likely isolated by Demerec and Fano
(1945) from a phage mixture provided by Dr Tony L Rakieten,(1) The
Long Island College of Medicine. The original source is speculated to be
from a lysate inoculated with raw sewage.
T4 contains a 168 kb dsDNA genome. When added to a sample,
T4 adds a known amount of DNA. The T4 DNA can then be extracted,
amplified, and detected as a control. T4 controls for the efficiency of
DNA extraction, the presence of PCR amplification inhibitors, the presence of intact
amplification reagents (Taq, buffer, dNTPs), and instrument functions (thermal cycling
and fluorescence detection).
Primers, probe, and master mix are available for amplification and detection of T4
DNA. The T4 primers are specific for a 163 bp portion of the tail tube glycoprotein 18
gene. The T4 detection probe is a molecular beacon. (2)

Extraction / amplification control with T4 bacteriophage
Adding T4 bacteriophage (BAC130) to the sample provides DNA for extraction,
amplification, and reaction condition PCR controls.
When added to a sample, T4 adds a known amount of DNA. The T4 DNA can
then be extracted, amplified, and detected as a control. T4 controls for the efficiency of
DNA extraction, the presence of PCR amplification inhibitors, intact amplification
reagents ( DNA polymerase, buffer, dNTPs), and instrument functions (thermal cycling
and fluorescence detection).
The T4 DNA may be detected in a separate PCR reaction using FAM labeled T4
probe ( PP100). Or the T4 DNA and test organism DNA may be detected using a
multiplex reaction using Quasar 670 labeled T4 probe (PP160) and FAM labeled test
organism probe).
Brief procedure for use of T4 as extraction and amplification control:
o Add 5µl bacteriophage to the sample. Proceed with DNA extraction.
o Dilutions of the bacteriophage may be made to give a final PCR Ct value that is
about 35. At this dilution, the phage is more sensitive, i.e. more likely, to detect a
poor extraction or the presence of PCR inhibitors in the reaction. If multiplex
reactions are being used, this dilution is less likely to compete with the test
organism PCR reaction.
Use of the T4 plasmid:
o Dilute the plasmid in TE to prepare a standard curve. Common dilutions would
be 10-fold from 200 to 0.02pg/ml. The 0.02 pg/ml plasmid dilution contains 12
copies of plasmid in 2 µl.

Detection of T4 DNA:
Attostar Master T4 master mixes
These master mixes require the addition of AmpliTaq Gold polymerase.
(available from Applied Biosystems, Part number AmpliTaq Gold LD. Obtain from
Applied Biosystems. Catalog number 4338857(1000 units) or 438856 (250 units).
https://www2.appliedbiosystems.com/
………………………………………………………………………………………………
Thermal cycle conditions for PCR reactions on RotorGene*
*Similar cycle conditions and reaction volumes may be used on many other
thermal cyclers.
95C 600 sec (activation for AmpliTaq Gold polymerase)
40 cycles
95C 15 seconds
60C 30 seconds RotorGene Channel Setup FAM/Sybr, Cy5; Gain 7
72C 30 seconds
FAM/Sybr has a source of 470nm and Detector 510nm (LightCycler use F1)
Cy5 has a source of 625nm and Detector 660hp nm
Quasar 670 has the same fluorescent absorption and emission as Cy5.
……………………………………………………………………………………………
Thermal cycle conditions for PCR reactions on LightCycler
95C 600 sec (activation for AmpliTaq Gold polymerase)
40 cycles
95C 15 seconds
60C 30 seconds acquire fluorescent signal on F1 gain =1
72C 30 seconds
40C 30 seconds cool

Attostar primers probe detection
These primer/probe reagents are used with AttoMaster 2X Mix
(Product number-AM10). AttoMaster 2X Mix contains Taq polymerase (requires heat
activation), dNTPs (0.4 mM) with optimal dUTP to dTTP ratio, heat labile UDG,
Mg (6 mM), and buffer.
……………………………………………………………………………………………
Thermal cycle conditions for PCR reactions on RotorGene*
*Similar cycle conditions and reaction volumes may be used on many other
thermal cyclers.

25ºC 10 min (UDG treatment time)
95ºC 120 sec (activation for AttoMaster polymerase)
40 cycles
95ºC 15 seconds
60ºC 30 seconds RotorGene Channel Setup FAM/Sybr, Cy5; Gain 7
72ºC 30 seconds
FAM/Sybr has a source of 470nm and Detector 510nm (LightCycler use F1)
Cy5 has a source of 625nm and Detector 660hp nm
Quasar 670 has the same fluorescent absorption and emission as Cy5.
………………………………………………………………………………………………
Thermal cycle conditions for PCR reactions on LightCycler

25ºC 10 min (UDG treatment time)
95ºC 120 sec (activation for AttoMaster polymerase)
40 cycles
95ºC 15 seconds
60ºC 30 seconds acquire fluorescent signal on F1 gain =1
72ºC 30 seconds
40ºC 30 seconds cool