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Buffers and Pipetting

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Name: Anarmaa Purevdorj

Date: 22 September 2017

Lab section: 803

Group 5

Partner: Samuel Jackson

Instructor: Ekaterina Zagriadskaia

Lab#1 Buffers and Pipetting

Introduction

The pipette is a very common equipment that is commonly used in forensic lab, research lab and biology lab. The pipet uses a vacuum to hold and draw the liquid which are called air displacement micropipettes. Most micropipettes are adjustable depending on their sizes and it ranges from 0.1µl-1000µL. Sizes fall down to P10 (0.5-10) P20 (2-20 µl), P200 (20-200), P1000(200-1000) and P5000(1000-5000 µl). P20 and P200 are the most common sizes that are mostly used in the lab.

Tris, KCl and LB (Luria Bertani) broth were prepared in this lab. Tris is one of the most common buffers that are used in biochemistry industry and mostly used to increase membrane permeability of the cell membranes. LB broth is mostly used for growth of bacteria and KCl (Potassium Chloride is an odorless and colorless appearance that used as a fertilizer in most fields.

Materials and Method

P200 and P20 pipetmen were used to draw 100µl and 10 µl of water. The water was dispensed into a weigh boat and the weight was recorded. Then process was repeated for three trials with 50% glycerol. The pipetman was kept to 100µl, but this time the plunger was pushed down to second stop before drawing up the water or 50% glycerol. Same procedure was repeated with P20 pipetman set to 10µl for three trials.

[pic 1]

was used to calculate the percentage difference.

100mM Tris, 500mM HCl and 500mL of LB broth (Luria Broth) were prepared. 1.218g of Tris was added to 75ml of distilled water. 1M of HCl was used to drop the pH from 10.25 to 7.9 and 10.6 mL of water was added to the solution to bring up volume to 100mL. 3.771g of KCl was added to 75ml of water. After dissolved, 24 mL of water added to the solution to bring up the volume to 100mL. 5.002g of Bacto-tryphone, 2.497g of yeast extraction and 5g of NaCl was added to 400mL of water. Then NaOH was added to the solution to adjust the pH to 7.49.

Result

Micropipette

Volume (μL)

Plunger Position

Expected Weight (g)

Experimental Weight (g)

% Difference

Average % Difference

P200 Water

100

1st Stop

0.1

0.1037

3.7

2.97

0.1014

1.4

0.0962

3.8

2nd Stop

0.1

0.1462

46.2

45.00

0.1437

43.7

0.1451

45.1

P20 water

10

1st Stop

0.01

0.0090

10.00

7.3

0.0102

2.00

0.0090

10.00

2nd Stop

0.01

0.0144

44.00

40.33

0.0141

41.00

0.0136

36.00

P200 Glycerol

100

1st Stop

0.113

0.1144

1.24

2.78

0.1148

1.6

0.1163

2.5

2nd Stop

0.113

0.1639

45.04

43.62

0.1662

40.00

0.1648

45.84

P20 Glycerol

10

1st Stop

0.0113

0.0131

16.00

15.05

0.0130

15.00

0.0129

14.15

2nd Stop

0.0113

0.0158

39.82

36.28

0.0158

39.82

0.0146

29.20

Table 1 Data collected for P200 and P20

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