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Bio Lab 4 Flowchart Lab 4

This document provides instructions for a biology lab experiment involving using micropipettes to transfer samples, centrifuging samples to separate cells from solution, extracting DNA from samples, and performing PCR on the extracted DNA samples. Key steps include using a P100 micropipette, centrifuging samples for 2 minutes, incubating DNA extracts at 75C for 15 minutes, and running PCR for 30 cycles.

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King Hunter12
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22 views

Bio Lab 4 Flowchart Lab 4

This document provides instructions for a biology lab experiment involving using micropipettes to transfer samples, centrifuging samples to separate cells from solution, extracting DNA from samples, and performing PCR on the extracted DNA samples. Key steps include using a P100 micropipette, centrifuging samples for 2 minutes, incubating DNA extracts at 75C for 15 minutes, and running PCR for 30 cycles.

Uploaded by

King Hunter12
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Bio Lab 4 Flowchart - LAB 4

Introductory to biology I (York University)

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Protocol Step Purpose of Step 1,4,5,9,11
1) MIcropipettes are biotechnological lab tools 1)Micropipettes are made in a variety of sizes
used to accurately measure small quantities of ,which allows researchers to select the optimal
liquids. It’s main components include: plunger, size for the volumes that need to dispense. What
, volume adjustment knob, are the most commonly used micropipettes and
volume indicator, , tip ejector arm, what is the volume range of the micropipette used
, and disposable tip. for this lab?(circle one)
P10,P20,P100,P200,P1000
2) Firstly, you need to get familiar with the P5,P15,P50, P300, P1500
micropipette as your experiment’s quality is P20,P40,P200,P400,P2000
dependent on your knowledge of use, as you
must handle the pipette holding it with one
hand fingers wrapped around the shaft with
your thumb positioned to push down on the
plunger.

3) When pushing down on the plunger you will


notice two “stops” which you should slowly
depressing the plunger until you feel the first
stop followed by doing the same process for
the 2nd stop.

4) Since in the lab you will be using a P100 4)If the micropipette’s volume indicator show the
micropipette you must make sure you set it to numbers 5, 6, to indicate a 56µL, what numbers
its correct volume via the volume adjustment should be shown when adjustmenting the
knob with, you being given visual micropipette to 40µL and 100µL?(circle one)
representation via the . 04 and 010, 40 and 100, 4 and 10

5) When collecting a sample using the 5)You should raise the plunger gently as if you
micropipette you must never use it without raise it too fast, the liquid may touch the end of the
adding the . When micropipette which results in the micropipette and
collecting you hold the micropipette in one your samples being (circle one).
hand, and the sample in the other as you Contaminated, the reason the sample will
should not have your lab partner hold the tube suddenly synthesise life, unusable as the samples
or pipette the sample in the sample rack. used afterwards would die due to the mixing of
samples.
6) As you get your sample pushed down and hold
the plunger at its first stop before lowering
mm of the tip into the solution then,
gently raise the plunger again to collect the
sample.

7) After filling the disposable tip, wait a second


and remove the tip to then check if there is any
air trapped in the tip and/or drops of
liquid outside the tip which would result in
incorrect volumes used in the experiment. You
would need to repeat steps - to make
sure none of these errors occur.

8) If no air bubbles are present inside the tip or


droplets outside of the tip check how much
liquid you have in the tip. However, you should
never set down your micropipette as liquid
from the tip may run into the
itself.

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9) Swab one are inside your cheek 3 times to 9)During the 30 seconds the swab is submerging
then dip the swab in a 1.5mL microtube within the PBS, the PBS changes from a (circle
that contains 400µL of one) to then become (circle one) as cells are
(PBS) then rinsed off the swab.
swirl the swab in the PBS for 30 seconds. brown,white clear, cloudy blue,red
You then press the swab against the inside of
the tube to ensure no PBS remains in the
swab.

10) Close the microtube and put your name on it


so your can add it to the centrifuge. Leave it
into the centrifuge at full speed for 2 minutes
which will collect the from the swab to
the microtube’s bottom.
11) You use the micropipette to slowly remove the
11) Once the centrifugation step is completed, PBS while disturbing the pellet via (circle one)
collect your sample to then remove the PBS in with the micropipette to prevent damaging the
the microtube without disturbing the pellet and having to restart the process.
of cells at the bottom of the microtube. Tickling, Touching, Scratching
12) After mixing the pellet solution you must use
12) Once the PBS is removed, add 20µL of 0.2M the heating block to incubate the sample for
NaOH solution into the microtube and mix (circle one) minutes at (circle one)oC.
for Ten, 75 fifteen, 50 five, 105
seconds or until the pellet completely
dissolves using a vortex.

13) Remove the microtube from incubation and


add 180µL of 10mM Tris-HCl(pH )
solution to neutralise the extraction. Gently tap
the microtube with your finger to mix the
solution then, place the microtube with the
extracted DNA on ice in preparation for the
PCR reaction step. 14) The stock solutions are labelled this way to
ensure the PCR solutions are not mixed and
14) For the PCR reaction step once at the fume make sure you use a new pipette tip between
hood where you add the 15µL of Blue reaction samples.
stock solution to a 0.2mL PCR tube(labelled
with a dot) and 15µL of Brown
reaction solution to another 0.2mL tube(with 15) The order of PCR conditions set by the
no dot). thermocycler are: 95oC for 2 minutes, 95oC for 30
seconds, (circle one) for 30 seconds, 72oC for 45
15) After returning to your lab bench, add 10µL of seconds, Repeat steps 2-4 (circle one) times,
your extracted DNA to each reaction tube, 72oC for 3 minutes and, hold for (circle one)
using a new pipette tip for each reaction to indefinitely.
avoid . Close the 58.1oC,thirty,16oC
tube to then gently mix the tube by tapping 75oC,twenty,18oC
with your finger, you then give your labelled 95oC,ten,250K
tubes to the TA to be added to the PCR
machine

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