Biochemistry Practical

Dept.of Biochemistry

Zhihong Li ( 李志红 )

REGULATIONS OF PRACTICAL

Successful experiments usually depend on accurate managements. All students should comply with these regulations.

•1.The students should come into the lab on time. It is prohibit being absent, late or leaving earlier. Anyone who is late for 15 minutes or more should be thought as absenteeism.

2. It is necessary to put on the work clothes before entering the lab. Not permitted to come into if the students are wearing vest, short pants or slipper.

3. Keep quiet and neat in the lab. Any actions, which are not concerned with your experiment, should be avoided. 

4. Wash your hands in time after finished the experiment or touched the toxic chemicals. 

5. Discard solid wastes in the provided waste containers, not in the sink!  Rinse non-hazardous, water-soluble wastes such as small amounts of acids down the drain.

6. The students should know the content of practical, the objectives, principle and procedure in advance.

7. Carefully observe the experimental phenomena, record the experimental result and discuss the significance of the experiment.

8. The students should clean the lab, take back the apparatus and materials, treat with the trash, and leave off after the permission of the instructor.

9. Carefully and accurately operate the apparatus. It should be kept in good order after your finishing the operation. It should be compensated according to the cause that results in the damage of the apparatus.

10. The students should hand over the experimental reports to the instructor in time.

Content

1 Volume transfer, accuracy and precision

2 Spectrophotometry & plotting of calibration curve

3 Alkaline phophatase

4 Enzyme inhibition test

5 Gel chromotography

6 Serum glucose test

7 Final experimental exam

Volume transfer, accuracy and

precision

BIO - 01

PURPOSE

To learn how to handle pipettes and electronic balance.

To learn how to dispense properly.

To learn the accuracy and precision.

TECHNIQUES FOR USING PIPETTES （移液管）

1.Pipettes should be used only with Rubber Bulbs（吸耳球） , mouth pipetting should not be allowed.

2.While taking and dispensing with right (left) hand, use the finger of the other hand to support the pipettes so that the pipettes might not be trembling.

3. Rinse out the pipette with the fluid to be measured and fill it by suction, holding it vertically while adjusting liquid level to the calibrating line during delivery.

The lowest part of the meniscus, when it is sighted at eye level, should be level with calibration line on the pipette.

Right

4.The flow of liquid should be restricted when using volumetric pipettes and the tip should be touched to the inclined surface of the receiving container, until three seconds after the liquid has ceased to flow.

5. Serological pipettes are calibrated to the tip so they should be blown out （吹） . The pipette is first allowed to drain as usual and then liquid is blown out.

These pipettes are generally used in handling reagents and are considered not accurate enough to handle standard and sample.

micropipette

Multi-channel pipettesingle-channel pipette

FUNCTION OF THE MICROPIPETTE（加样枪）

• Micropipettes are Micropipettes are used to transfer small liquid used to transfer small liquid volumesvolumes..

• It is a precision instrument calibrated in microliters (μL). 1ml = 1000 (μL)

• It was invented in 1957 by Heinrich Schnitger iIt was invented in 1957 by Heinrich Schnitger in Germany.n Germany.

CAREFUL HANDLING OF THEMICROPIPETTE

• The micropipette must be handled with great care, to avoid damage.

• A damaged micropipette may produce imprecise measurements and this could affect the results of the experiment.

• Do not rough-handle or drop the micropipette.

Structure of micropipette•Push botton

• handle • tip ejector

• digital volume indicator

• tip ejector arm

• tip hold

ti p0.5-20µl

20-200µl100-1000µl

Select proper Pipette:

Micropipette have different volume range.

P5000   1,000 ~ 5,000μlP1000     200 ~ 1,000 μlP200      50 ~ 200 μlP100       20 ~ 100 μlP20        2 ~ 20 μlP10        0.5 ~ 10 μl

How to handle micropipette:

MicropipettesMicropipettes

• Micropipettes have Micropipettes have 3 positions3 positions::

– Rest position.Rest position.

– First stop. First stop.

– Second stop.Second stop.

Volume Adjustment Knob:

Digital Volume Indicator:

Pipettors – 3 Volumes:Step 1: Set and read the VolumeStep 1: Set and read the Volume

Operating the MicropipetteOperating the Micropipette

• Hold the micropipette in one hand and with the other hand, turn the volume adjustment knob (or shaft) until the volume indicator displays the desired volume.

• IF THE VOLUME ADJUSTMENT KNOB IS HARD TO TURN, STOP AND CALL THE INSTRUCTOR.

• DO NOT TRY TO FORCE THE KNOB TO TURN BECAUSE YOU WILL DAMAGE THE MICROPIPETTE.

• Do not adjust the micropipette volume above or below that recommended(!!!).

Example of tip sizes:

Attaching the disposable tip

Operating the Micropipette-2Operating the Micropipette-2

Step 2: Attach the Disposable TipStep 2: Attach the Disposable Tip• Select a box containing the correct size tips to be used.• Place the pointed end of the micropipette shaft into one of the tips. • Press down and twist slightly to ensure an airtight seal, then lift-up the micropipette with tip attached.•YOU MUST NEVER USE THE MICROPIPETTE WITHOUT A TIP ATTACHED !!!

Operating the Micropipette-3Operating the Micropipette-3

Step 3: Depress the Plunger to the First Stop

Step 5: Draw up the sample

Step 6: Pause

•To aspirate the sample into the tip, allow the pushbutton to return slowly and smoothly to the fully extended UP POSITION. NEVER LET THE PLUNGER SNAP UP!

•Wait a few seconds to ensure that the full volume of sample is drawn into the plastic tip. WAIT LONGER FOR LARGER VOLUMES. WAIT LONGER FOR MORE VISCOUS ("SYRUP-LIKE") SUBSTANCES.

Step 4: Immerse Tip in Sample

Operating the Micropipette-4Operating the Micropipette-4

Step 7: Withdraw the Tip

• Remove the tip from the sample liquid. No liquid should remain on the OUTSIDE of the tip. • Visually examine the tip to make sure it contains reagent and that there are no bubbles present.

 

Operating the Micropipette-5Operating the Micropipette-5Step 8: Dispense the Sample•To dispense the sample from the pipette:

a) Touch the tip end to the side wall of the receiving vessel. b) Depress the plunger to the FIRST STOP. c) Pause for at least one second .d) Press the plunger to the SECOND STOP (the second point, of greater resistance, at the bottom of the stroke) to expel any residual liquid in the tip (like "blowing out" a glass pipette).

                                                     

(a) Start Dispensing 

(b) 1st Stop = Dispense

(c) 2nd Stop = Expel

Operating the Micropipette-6Operating the Micropipette-6Step 9: Withdraw the Pipette•With the plunger fully depressed, withdraw the pipette from the receiving vessel carefully.

Step 10: Release the Plunger•Gently allow the plunger to return to the UP position. DO NOT allow it to SPRING BACK! Step 11: Discard the Tip•Discard the tip by depressing the tip ejector button, as shown below. A fresh tip should be used for each sample to prevent sample carryover.

Press ejector button to discard tip.

Step-wise Operation of the Step-wise Operation of the MicropipetteMicropipette

• Set volume.Set volume.• Attach disposable tip.Attach disposable tip.• Depress the plunger to Depress the plunger to 11stst stop stop..• Immerse tip in sample and Draw up Immerse tip in sample and Draw up

sample.sample.• Withdraw the tip.Withdraw the tip.• Dispense the sample by pushing the Dispense the sample by pushing the

plunger to the plunger to the 22ndnd stop stop..• Withdraw the pipette and release the Withdraw the pipette and release the

plunger.plunger.• Discard the tip.Discard the tip.

Pippetting Guidelines and PrecautionsPippetting Guidelines and PrecautionsFor optimal reproducibility, use the following pipettin

g procedures:

(1) Consistent SPEED and SMOOTHNESS when you press and release the PLUNGER

(2) Consistent pressure on the PLUNGER at the FIRST STOP

(3) Consistent and sufficient IMMERSION DEPTH

(4) Nearly VERTICAL POSITIONING of pipette

(5) AVOID ALL AIR BUBBLES: Since the plastic pipette shaft can be damaged if liquids are drawn beyond the

tip into the shaft.

(6) NEVER lay the pipette on its SIDE nor INVERT the pipette if liquid is in the tip.

Accuracy and PrecisionAccuracy and Precision

• In Science, we want measurements to be both accurate and precise.

• What is the difference between them?

Accuracy and PrecisionAccuracy and Precision

• Accuracy is a measure of rightness.– It means "capable of providing a correct reading or

measurement." – It refers to how closely a measured value agrees wi

th the actual value.• Precision is a measure of exactness.

– Precise means “repeatable, reliable, getting the same measurement each time.”

– It refers to how closely individual measurements agree with each other.

Three Three targets targets with three with three arrows arrows each to each to shoot.shoot.

Can you hit the bull's-eye?Can you hit the bull's-eye?

Both accurate and precise

Precise but not accurate

Neither accurate nor precise

How do How do they they compare?compare?

Absolute error : E = Xi - XT

Relative error :

(1) Accuracy and error

100%rT

EE

X

(2) precision and deviation standard deviation (SD or S)

Relative Standard Deviation (RSD) or Coefficient of Variation (CV)

%100X

SCV

Mean: X= n

xxx n 21

How to use electronic balance

• Operation – Switch on /power– Tare ,make scale “0.000”– Weight ： record the weight

• Attention •

Glass Pipette1) Prepare a electronic balance2) Prepare 200 ml of distilled water3) Prepare a glass beaker4) Weigh the glass beaker and Tare out the balance (i.e., make the scale 0.000)5 ） Select glass pipettes and rubber bulb6 ） Dispense 3.8ml (or 1.6ml) and read the balance and record the weight7) Repeat 3 times and record the weight8 ） Calculate the Volume using the recorded weight and Temp-Density conve

rsion table.9 ） Calculate : ⒈Mean of volumes transferred• ⒉ Standard Deviation of volumes

PRACTICE

1) Prepare a electronic chemical balance2) Prepare 200 ml of distilled water and measure the temperature3) Prepare a glass beaker (20 – 50 ml size)4) Weigh the glass beaker and Tear out the balance (i.e., make the scale 0.000)5) Dispense 550 µl (or 160 µl) and read the balance and record the weight6) Repeat 3 times and record the weight7) Calculate the Volume using the recorded weight and Temp-Density conversion table.8) Calculate : Mean ⒈ ⒉ Standard Deviation of volumes transferred

MicropipetteMicropipette

Results

PipetteTrue(ml)

3.8 (or 1.6)

Roll numbe

r

number

1 2 3 4 5 6 7 8 9 10

11

12

Test(g)Volum

e(ml)

Mean

SD

MicropipetteTrue(µl)

550 (or 160 )

Roll numbe

r

number

1 2 3 4 5 6 7 8 9 10

11

12

Test(g)

Volume

(µl)

Mean

SD

Volume (ml)= Mass (g) / Dense (g/cm3)

Table. the different density of water at different temperature

T(0C)Density(g/

cm3)T(0C) Density(g/cm3) T(0C)

Density(g/cm3)

0 0.999 868 12 0.999 525 24 0.997 326

1 0.999 927 13 0.999 404 25 0.997 074

2 0.999 968 14 0.999 271 26 0.996 813

3 0.999 992 15 0.999 126 27 0.996 542

4 1.000 000 16 0.998 970 28 0.996 262

5 0.999 992 17 0.998 802 29 0.995 973

6 0.999 968 18 0.998 623 30 0.995 676

7 0.999 929 19 0.998 433 31 0.995 369

8 0.999 876 20 0.998 232 32 0.995 054

9 0.999 869 21 0.998 021 33 0.994 731

10 0.999 728 22 0.997 799 34 0.994 399

11 0.999 632 23 0.997 567 35 0.994 059

Next experiment

• Spectrophotometry & Plotting of Calibration Curve (p11~16)