Water Content of a PCR Kit

Polymerase chain reaction (PCR) is a method frequently used in molecular biology. PCR rapidly produces replicas of a particular DNA segment. This segment is amplified exponentially to produce millions of duplicates.

A PCR kit is made up of the template DNA, the temperature-resistant DNA polymerase, the dNTPs (deoxynucleoside triphosphates), Mg2+, the primers and buffer salts. It can be obtained in liquid or dried form. The water content is a vital specification in the dried form relative to the appropriate storage of the PCR-kit.

This note details the coulometric Karl-Fischer titration of a dried PCR-kit utilizing the InMotion KF Pro for water gas-phase extraction attached to the C30S coulometric KF-titrator. The water content is anticipated to be <2%.

Below, the image shows a carrier foil with six individual PCR-kits immobilized on it. Two such kits were used for the analysis, which is relative to a sample size of ~ 0.012 g.

Image Credit: Mettler Toledo - Titration

Sample preparation and procedures

  1. Principle of gas-phase extraction: Nitrogen penetrates via an SV2 valve (inlet pressure should be 0.5 bar < P < 1.3) and enters a drying unit packed with silica gel, before passing through a drying unit filled with molecular sieve.
    The flow rate is regulated by a digital gas flow control (typical 80 – 150 mL/min) that is determined in the titration method. The dry air transports the water vapor from the sample and transports it into the titration cell.
  2. Blank preparation: Position the empty 10 mL blank vial on position 1 of the InMotion KF. Use the screw cap to close the vial.
  3. Sample preparation: Introduce ~ 0.012 g PCR-kit into a 10 mL vial and close it using the screw cap. Once all vials are filled, place into the respective positions on the InMotion KF rack.
  4. Start of analysis: Position an empty vial - closed with a screw cap - into the drift position on the rack. Commence M806. Initially, the method will carry out a pre-titration to eliminate the excess water from the titration cell and will then go into Standby mode.

Conduct manual drift determinations until the value of online drift is < 5 µg/min. Following this step, press ‘Start sample.’ Usually, the method will initiate with the subsequent sequence: Drift – Blank – Sample.

In most instances, the drift is decided once, prior to the sample loop, and this drift value is employed for all successive calculations.

Chemistry

Water, M = 18 g/mol.

ROH + SO2 + 3 RN + I2 + H2O ⇌ (RNH) • SO4 R + 2 (RNH)I

Solutions and chemicals

  • Sample: PCR-kit immobilized on carrier foil
  • Chemicals: Coulomat AG oven

Instruments and accessories

  • Compact line titrator C30 SX (30252662) or Titration Excellence T7 (30252675) or T9 (30252676)
  • InMotion KF-Pro (30407502)
  • LabX software
  • Sample vials 10 mL with screw caps (30407445, 30407436)
  • SV2 valve (30407442)
  • XPR205 Analytical balance (30355411)

Results and discussion

Scan of the PCR-kit sample from 50 to 250 °C

Scan of the PCR-kit sample from 50 to 250 °C. Image Credit: Mettler Toledo - Titration
 

Titration of the PCR - kit sample 2/6

Titration of the PCR - kit sample 2/6. Image Credit: Mettler Toledo - Titration

Table 1. Source: Mettler Toledo - Titration

R2: Water content
Blank: [µg], Sample: [%]
Blank 92.6
1 1.495
2 1.497
3 1.478
4 1.484
5 1.506
6 1.456
Mean
s
srel
1.481
0.261
1.766%

The repeatability and recovery are set between specifications. The average value of 1.481% is below 2%, as defined.

Remarks

Initially, the optimal gas-phase extraction temperature had to be established by conducting a scan of the sample. It was found that decomposition commenced above 200 °C.

160 °C was therefore selected as the gas-phase extraction temperature. To prevent oxidation of the organic substances present in the PCR-kit for this biological sample, nitrogen is used as the carrier gas.

Waste disposal and safety measures

Dispose of as an organic solvent.

References

Good titration practice in Karl-Fischer Titration (30245921)

Measured data

Table 2. Table of measured values – PCR kit sample 2/6. Source: Mettler Toledo - Titration

C [mC] E [mV] Drift [µg/min] H2O [µg] t [s]
0 169 0 0 0
78.3 177.7 95.8 7.3 1
211.1 166.1 648 19.7 2
293.6 150.9 660.9 27.4 3
362.3 132.2 681.5 33.8 4
398.2 124.3 514.2 37.2 5
428.1 127.8 388.6 40 6
465.5 122.9 351.2 43.5 7
502.8 120.8 330.3 46.9 8
537.2 115.1 272.8 50.1 9
3176.9 97 0 296.6 591
3176.9 99.5 0 296.6 592
3176.9 101.6 0 296.6 593
3177 98.7 0 296.6 594
3177 99.1 0 296.6 595
3177 101.8 0 296.6 596
3177.2 98.6 0 296.6 597
3177.2 98.2 0 296.6 598
3177.2 96.6 0 296.6 599
3177.2 96.1 0 296.6 600
3177.2 102.4 0 296.6 600

About Mettler Toledo - Titration

METTLER TOLEDO instruments are used in research, scientific and quality control labs, amongst many others in the pharmaceutical, chemical, food and cosmetics industries. They are a global market leader with the three instrument groups most frequently used in the laboratory, like balances, pipettes, and pH meters, and we are a pioneer in the field of Automated Chemistry.

METTLER TOLEDO industrial solutions cover the diverse steps in a host of manufacturing processes at many of the same customers as served for the laboratory. Solutions range from receiving raw materials, in-line process control and end-of-line packaging control, to logistics and shipping. Increasingly, these solutions are fully integrated into the customer's IT environment, helping automate their workflows.


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Last updated: Jan 22, 2021 at 8:42 AM

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