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 1Volume transfer, accuracy and precision 2 Spectrophotometry & plotting of calibration curve 3Alkaline phophatase 4Enzyme inhibition test 5Gel chromotography 6Serum glucose test 7Final 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 pipette single-channel pipette

FUNCTION OF THE MICROPIPETTE （加样枪） Micropipettes are used to transfer small liquid volumes.Micropipettes are used to transfer small liquid volumes. It is a precision instrument calibrated in microliters (μL). 1ml = 1000 (μL) It was invented in 1957 by Heinrich Schnitger in Germany.It was invented in 1957 by Heinrich Schnitger in Germany.

CAREFUL HANDLING OF THE MICROPIPETTE 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

Select proper Pipette: Micropipette have different volume range. P5000 1,000 ~ 5,000μl P ~ 1,000 μl P ~ 200 μl P ~ 100 μl P20 2 ~ 20 μl P ~ 10 μl How to handle micropipette:

Micropipettes Micropipettes have 3 positions:Micropipettes have 3 positions: –Rest position. –First stop. –Second stop.

Volume Adjustment Knob: Digital Volume Indicator: Pipettors – 3 Volumes: Step 1: Set and read the Volume Operating 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-2 Step 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-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-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-5 Step 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-6 Step 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 Micropipette Step-wise Operation of the Micropipette Set volume.Set volume. Attach disposable tip.Attach disposable tip. Depress the plunger to 1 st stop.Depress the plunger to 1 st stop. Immerse tip in sample and Draw up sample.Immerse tip in sample and Draw up sample. Withdraw the tip.Withdraw the tip. Dispense the sample by pushing the plunger to the 2 nd stop.Dispense the sample by pushing the plunger to the 2 nd stop. Withdraw the pipette and release the plunger.Withdraw the pipette and release the plunger. Discard the tip.Discard the tip.

Pippetting Guidelines and Precautions For optimal reproducibility, use the following pipetting 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 Precision In Science, we want measurements to be both accurate and precise. What is the difference between them?

Accuracy 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 with 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 targets with three arrows each to shoot. Can you hit the bull's-eye? Both accurate and precise Precise but not accurate Neither accurate nor precise How do they compare?

Absolute error : E = X i - X T Relative error : (1) Accuracy and error

(2) precision and deviation standard deviation (SD or S) Relative Standard Deviation (RSD) or Coefficient of Variation (CV) Mean: X=

How to use electronic balance Operation –Switch on /power –Tare,make scale “0.000” –Weight ： record the weight Attention

Glass Pipette 1) Prepare a electronic balance 2) Prepare 200 ml of distilled water 3) Prepare a glass beaker 4) Weigh the glass beaker and Tare out the balance (i.e., make the scale 0.000) 5 ） Select glass pipettes and rubber bulb 6 ） Dispense 3.8ml (or 1.6ml) and read the balance and record the weight 7) Repeat 3 times and record the weight 8 ） Calculate the Volume using the recorded weight and Temp-Density conversion table. 9 ） Calculate : ⒈ Mean of volumes transferred ⒉ Standard Deviation of volumes PRACTICE

1) Prepare a electronic chemical balance 2) Prepare 200 ml of distilled water and measure the temperature 3) 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 weight 6) Repeat 3 times and record the weight 7) Calculate the Volume using the recorded weight and Temp- Density conversion table. 8) Calculate : ⒈ Mean ⒉ Standard Deviation of volumes transferred Micropipette

Results Pipette True (ml) 3.8 (or 1.6) Roll number number Test(g) Volume (ml) Mean SD Micropipette True (µl) 550 (or 160 ) Roll number number Test(g) Volume (µl) Mean SD Volume (ml)= Mass (g) / Dense (g/cm 3 )

Table. the different density of water at different temperature T( 0 C)Density(g/cm 3 )T( 0 C)Density(g/cm 3 )T( 0 C)Density(g/cm 3 )

Next experiment Spectrophotometry & Plotting of Calibration Curve (p11~16)