basic molecular biology techniques - DNA and RNA isolation from plant sample, nanodrop technique, pcr and cloning.

1. Presented by- Ravi Ranjan

2. Experiments
performed:-
DNA isolation from Jatropha curcas plant sample
RNA isolation form Jatropha curcas plant sample
Determination of quality and quantity of isolated DNA and RNA
Polymerase chain reaction (PCR)
Cloning of Amplified fragment :-
Isolation of plasmid DNA
Ligation
Competent cell preparation
Transformation
Screening
Recombinant plasmid isolation

3. Jatropha curcas
Jatropha is poisonous, semi evergreen shrub
(6m tall).
They are also grown in deserts.
Seeds of jatropha are rich in oil, which might
be a new source of bio fuel.
DNA or RNA isolation was performed using
the leaf of the same plant.
In cloning also the genes of this pant was
used ( DGAT, PAP, LPAT, GPAT) which are
involved in tri-acyl glycerol pathway (Bio-
Diesel production pathway).

4. DNA ISOLATION BY
C TAB METHOD
DNA is a higher molecular weight
molecule and can be isolated from
plant tissue by fracturing cell wall,
and cell membrane using a
detergent and it can be purified
from other contaminants using
P:C:I.
The isolation of plant nucleic acid
is a fundamental requirement for
most characterization, for
identification and isolation of
genes from genetic material.
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment

5. C TAB method of DNA isolation
Leaf sample is taken, grinded, and taken in micro-centrifuge tube,
pre-warmed C TAB is added, and RNase.
Incubated at 66˚C for 1 hour.
C:I added, mixed, centrifuged at 1000rpm for 10 minutes, aqueous
phase is transferred to another tube.
Isopropanol and sodium acetate is added and mixed well, kept at
20 ˚C for 1 hour or overnight.
Centrifuged at 10000rpm for 10 minutes, pellet is retained and
washed using ethanol.

6. RNA ISOLATION BY
C TAB METHOD
RNA can be isolated from plant
tissue by C TAB method.
The isolation of RNA is
preferred over DNA in genomic
laboratories or banks, since
cDNA synthesis and library
preparation is easy to maintain
because it doesn’t have interons.
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment

7. C TAB method of RNA isolation
Leaf sample is taken, grinded, and taken in micro-centrifuge tube, extraction
buffer is added, and RNase.
Incubated at 65˚C for 30 minutes.
C:I added, mixed, centrifuged at 12’000rpm for 10 minutes, aqueous phase is
transferred to another tube.
Upper phase was taken, C:I is added again centrifuged at 1000rpm.
Aqueous phase is taken, Isopropanol is added and centrifuged at 12’000 rpm .
Pellet is washed using ethanol (centrifuged at 12’000rpm) and air dried, and
retained in nuclease free water.

8. Determination of quality and quantity of
isolated
DNA and RNA
The quality and quantity of the genomic
DNA/RNA was checked using AGE. Good
quality of DNA/RNA shows intact band
without smearing.
The purity and concentration
of DNA/RNA was checked
using NANODROP
spectrophotometer at OD
260/280 taken against TE or
nuclease free water as blank.
The DNA or RNA sample showing the OD
260/280 value between 1.7-1.9 was
considered as pure sample and the
concentration of genomic DNA was
estimated.
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment

9. Casting of Agarose gel
 The casting unit was setup.
 0.8% Agarose DNA, and 1.2% RNA Agarose is dissolved in
100ml of 1x TAE.
 Heated to dissolve and 4µl of EtBr is added before
cooling.
 Poured in tray and comb was placed.
 Left for solidification at room temp. for 30 minutes.

11. DNA and RNA bands observed in UV Transilluminator

12. Polymerase chain reaction (PCR)
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
A biochemical technology used to amplify a
single or a few copies of a piece of DNA across
several orders of magnitude, generating
thousands or millions of copies of a particular
DNA sequence.
The process mainly involves three basic stages:
Denaturation: at 94-96˚C, the ds DNA
denatures into single separated strands.
Annealing: at 50-65˚C, two primers
anneal the complementary seq. on either
side of DNA.
Extension: at 70-72˚C, the polymerase
binds to and extends the complementary
DNA stand from each primer.

13. 1.
• Denaturation
2.
• Annealing
3.
• Extension
3.
Primary Denaturation
Final Extension
X 35

14. Cloning
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
CLONE: A collection of molecules or cells which
are all identical to an original molecule and cell.
CLONING VECTOR: It is a DNA molecule in
which foreign DNA can be inserted and which is
capable of replicating within host cell to
produce multiple clones of recombinant DNA.
CLONING STEPS:-
Plasmid isolation
Ligation
Competent cell preparation
Transformation
Screening (blue white screening)
Recombinant plasmid isolation

16. Isolation of plasmid DNA
(from E. coli)
The cells are centrifuged and, resuspended pellet in different
buffers(STE buffer, GTE buffer)after each centrifuge.
Lysozyme solution is added, mixed.
Solution ii and iii is added (double volume and 1/3rd)and incubated at
room temperature and ice respectively, and again centrifuged.
Supernatant is taken in fresh tube, added alcohol and refrigerated
overnight.
Centrifuged, washed with ethanol and dried.
Dissolved in TE buffer so that it can be used further.

17. Ligation
Reagents Volume used
5X ligation buffer 2µl
PGEM-T vector 1µl
T4 ligase 1µl
Sterile water Add to final volume of 10µl
Fresh PCR product 2-3µl
Final volume 10µl
The reaction was mixed gently and incubated at 4˚C for overnight.

18. Competent cell preparation
Single colony is picked up and grown overnight.
Pre-warmed algae media is added and stored at 37˚C, cooled
on ice and transferred to sterile centrifuge tube.
Centrifuge at 4000g for 5 min at 4 ˚C, supernatant is discarded
n tube is kept on ice.
Resuspended in 30 ml solution i containing RbCl2, KCH2COOH,
CaCl2 and glycerol for 90 minutes, and centrifuged it again and
pellet resuspended in 4 ml solution containing MOPS, RbCl2
e.tc.

19. Transformation
competent cells and DNA is added in eppendroff tubes, stored in ice for 3o
minutes, and then transferred to pre-heated water bath at 42˚C for 90
seconds.
Tubes are immediately transferred to ice box, and kept for 2 minutes.
incubated in water bath at 37˚C for 45 minutes, and is kept in shaker
incubator for 15 minutes.
Transformed cells are transferred on Petri plates containing algae agar
medium, and spread, incubated at 37˚C for 18 hours.

20. Screening
+ Amp

21. Recombinant plasmid isolation
white colonies are picked and inoculated in 3 ml LB broth and incubated
at 37 ˚C for overnight in shaker.
Cultures is centrifuged for 1 minute at 14000 rpm, and resuspended in
Tris and EDTA containing solution.
solution with NaOH, SDS, potassium acetate is added and mixed gently, a
white precipitate is observed.
Tube is centrifuged for 10 minutes at 14000rpm and supernatant is taken
in fresh tube.
Ethanol washing is done, air dried and suspended in sterile nuclease free
water or TE buffer and stored.