BIOLOGY-1

LAB REPORT NO. 04

Dept. of Civil Engineering

 Objectives:
To learn how DNA can be extracted from blood sample.

 Theoretical explanation:
DNA from human blood is extracted for variety of purposes such as
parenting, crime detection etc. This experiment will help us understand the process by
performing different experimental steps.

 Apparatus and Chemical:

 RBC lysis buffers
 Centrifuge machines
 WBC lysis buffers
 Proteinase K
 TE Buffer.

 Procedure:
 Take a sample of blood.
 Take 760 micro liter blood was added in an Eppendorf and an equal volume of RBC lysis
buffer.
 RBC lysis buffer and blood were mixed into each other by turning up and down the
Eppendorf and at room temperature, the sample was incubated
 After incubating the sample, it was centrifuged in the micro centrifuge as 13,000 rpm
(revolutions per minute) for I minute.
 Discard the supernatant and add 500µl RBC buffer.
 Sample was again centrifuged at 13,000 rpm and minute.
 Now again supernatant was discarded and 500micro WBC lysis buffer was added in the
pellet. Along with solution Sol. 2, 20% SDS (I4pl) and proteinase K (12pl) were also
 added. The pellet was dissolved in the solution and all the samples were incubated in the
incubator for overnight period et 37C.
 On second day, solution C and solution D were prepared individually and then mixed
both the solutions in equal properties.
 In the Eppendorf, upper and lower layer was formed after centrifuge. The upper layer was
taken and shifted into the other Eppendorf and lower layer was discarded.
 Chilled isopropanol with the volume of 500pl and chilled sodium acetate with the volume
of 55pl were added and mixed them well be overturning the Eppendorf several times.
Now at the step, DNA was precipitated out.
 The sample was again centrifuged at 13,000 rpm and for 10 minutes. After centrifugation,
the pellet appeared at the bottom of Eppendorf
 At that step, the supernatant was discarded and then 70% of ethanol with the volume of
200;11 was added for washing. After adding ethanol, the sample was centrifuged at
13,000 rpm and for 7 minutes.
 Again, supernatant was discarded as it contains the ethanol and pellet was dried out after
placing the Eppendorf in the incubator for almost 2 hours.
 After 2 hours, T.E buffer with the volume of 1500 was added 0 he Eppendorf and dried
pellet was dissolved in it
 Now place the Eppendorf in the incubator for overnight incubation at 37C temperature.
 On the next day, sample was taken out from the incubator and for checking the quality
and quantity of that extracted DNA within the sample, 1% own50 gel was run the
analysis W. done at the gel DOC system by UV
Trans-illuminator.

 Results and conclusions:

Bands of the extracted DAN can be seen under the UV; Thickness of the band
will indicate the quantity of the DNA.

 Precautions:
 Before starting, it is always important to ensure that the working surface is clean
and that you are wearing a pair of clean gloves to avoid contamination.
 Discard the tips properly.
 After completing experiment clean the working surface with 70% ethanol.
 Wash your hands with sanitizer.