SCREENING OF AEQUORIN TRANSGENICS OF CHICKPEA

HANDS ON TRANING REPORT

Submitted by

HARSH
Under the Supervision of

DR. MUKESH KUMAR MEENA

Scientist- II

National Institute of Plant Genome Research

In fulfilment of two months hands on traning

DEPARTMENT OF BIOTECHNOLOGY

DEENBANDHU CHOTTU RAM UNIVERSITY OF SCIENCE AND

TECHNOLOGY

MURTHAL-HARYANA

1
 ACKNOWLEDGEMENT
First of all, it is my great pleasure to pay my sincere thanks and heartiest gratitude to Prof. Kiran Nehra,
Head of Department of Biotechnology, University of Haryana, for providing me all the necessary
permissions and valuable help so that I can carry out my hands on traning efficiently.
Sincere thanks to the Academic department of the National Institute of Plant Genome Research, New
Delhi for giving me the opportunity for carrying out my traning.
I wish to express my gratitude to my guide Dr. Mukesh Kumar Meena, Scientist II, National Institute of
Plant Genome Research, New Delhi for being an inspiring guide, his outstanding cooperation, constant
encouragement and dedication in my traning.
Last but not the least; I would like to end my acknowledgement by thanking my supportive friends and
my family members for being the main source of my energy, support and encouragement throughout my
life.

LIST OF FIGURES

Caption Page
Number

Serial Protocol for extraction of genomic DNA. 4

2
Number

1.

2. Protocol for genomic DNA extraction. 5

3. Introduction to PCR. 6

4. Plasmid Map. 7

5. Screening of chickpea Aequorin lines by Aeq 8-18

PCR.

3
Genomic DNA Extraction In 1.5ml Eppendorf
Extraction Buffer (EB)-
0.2M Tris-HCL (pH-8)
0.4M LiCl
25mM EDTA
1% SDS
Stock- For 50ml
1M Tris-HCL (pH-8) 10ml
2M LiCl 10ml
0.5M EDTA (pH-8) 2.5ml
10% SDS 5ml
dH2O 22.5ml
Procedure-
Collect small amount of tissue (1 young 0.5x0.5cm leaf).
Add liquid nitrogen (-196℃) to each tube.
Crush the tissue with micropestals until tissue become fine powder.
Add 300uL EB and centrifuge at maximum rpm (13000) for 10 minutes.
Transfer the Supernatant to a new labelled tube and add 250uL ice- cold Isopropanol.
Centrifuge at maximum rpm for 15 minutes to pellet DNA and pour off supernatant.
Add 70% Ethanol and invert centrifuge at maximum rpm for 10 minutes.
Pour off Ethanol and air dry the tube.
Resuspend in 70uL autoclaved ddH2O.
Perform PCR for screening of Aeq transgenics in chickpea.

4
Polymerase Chain Reaction (PCR)
PCR developed by Kary Mullis in the 1980s. PCR is based on using the ability of
DNA polymerase to synthesize new strand of DNA complementary to the offered
template strand. Because DNA polymerase can add a nucleotide only onto a
preexisting 3'-OH group, it needs primer to which it can add the first nucleotide.

Components of PCR:

DNA template:
DNA that contains the target sequence. At the beginning of the reaction, high
temperature is applied to the original double-stranded DNA molecule to separate the strands
from each other.

DNA polymerase:

The enzyme that synthesizes new strands of DNA complementary to the target sequence. The
first and most commonly used of these enzymes is TaqDNA polymerase (fromThermis
aquaticus), whereas PfuDNA polymerase (fromPyrococcus furiosus) is used widely because of
its higher fidelity when copying DNA. Although these enzymes are subtly different, they both
have two capabilities that make them suitable for PCR:
1) They can generate new strands of DNA using a DNA template and primers.
2) They are heat resistant.

Primers:

Short pieces of single-stranded DNA that are complementary to the target sequence. The
polymerase begins synthesizing new DNA from the end of the primer.

Nucleotides (dNTPs or deoxynucleotide triphosphates):

Single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.

PCR Program:

5
Plasmid map (pBl121- Aequorin)
NOS-pro: Nopaline synthase promoter
NOS-ter: Nopaline synthase terminator
CaMV 35s: Cauliflower Mosaic Virus promoter
NPTII: Neomycin phosphotransferase II

6
AIM- Screening of chickpea Aequorin lines by Aeq PCR.
PCR Stages-
Stage 1
Primary Denaturation- 98℃ for 30 second
Stage 2 (35 Rounds)
Secondary Denaturation- 98℃ for 10 second
Annealing- 55℃ for 30 second
Amplification- 72℃ for 30 second
Stage 3
Last Amplification- 72℃ for 5 minutes
Infinite Hold- 4℃
Aeq Product- 295 bp
Control Primer PCR- 200 bp (Sense F+ Sense R)
Total Reaction- 6
Total Reaction Volume- 10uL

PCR Standard Total Working

Components volume Reaction Volume(uL)
(uL)
GT master mix 5 6 30
10uM Primer F 0.25 6 1.5
10uM Primer R 0.25 6 1.5
Genomic DNA 1 6 6
MQ water 3.5 6 21

7
RESULT- Control PCR worked well for all 6 DNA samples whereas NO PCR with Aequorin
Primers.
Inference- PCR program worked well.

8
AIM- Screening of chickpea Aequorin lines by Aeq PCR.
PCR Stages-
Stage 1
Primary Denaturation- 98℃ for 30 second
Stage 2 (35 Rounds)
Secondary Denaturation- 98℃ for 10 second
Annealing- 55℃ for 30 second
Amplification- 72℃ for 30 second
Stage 3
Last Amplification- 72℃ for 5 minutes
Infinite Hold- 4℃
Aequorin Product- 295 bp
PCR Elements-
Total Reaction- 13
Total Reaction Volume- 10uL
Transgenic Plant sample- 11, Wild Type- 1, Aequorin Plasmid- 1

PCR Standard Total Reaction Working

Components volume (uL) Volume(uL)
GT master mix 5 13 65
10uM Primer F 0.25 13 3.25
10uM Primer R 0.25 13 3.25
Genomic DNA 1 13 13
MQ water 3.5 13 45

9
RESULT- NO band in sample, band only shown in Positive control.
Inference- NO plants carry Aequorin plasmid.

10
AIM- Screening of chickpea Aequorin lines by Aeq PCR.
PCR Stages-
Stage 1
Primary Denaturation- 98℃ for 30 second
Stage 2 (35 Rounds)
Secondary Denaturation- 98℃ for 10 second
Annealing- 55℃ for 30 second
Amplification- 72℃ for 30 second
Stage 3
Last Amplification- 72℃ for 5 minutes
Infinite Hold- 4℃
Aequorin Product- 295 bp
PCR Elements-
Total Reaction- 30
Total Reaction Volume- 10uL
Transgenic Plant sample- 13-40, Aequorin Plasmid- 1

PCR Standard Total Working

Components volume Reaction Volume(uL)
(uL)
GT master mix 5 30 150
10uM Primer F 0.25 30 7.5
10uM Primer R 0.25 30 7.5
Genomic DNA 1 30 30
MQ water 3.5 30 105

11
RESULT- All samples were NEGATIVE, Positive control amplified.
Inference- NO plants carry Aequorin plasmid.

12
AIM- Screening of chickpea Aequorin lines by Aeq PCR.
PCR Stages-
Stage 1
Primary Denaturation- 98℃ for 30 second
Stage 2 (35 Rounds)
Secondary Denaturation- 98℃ for 10 second
Annealing- 55℃ for 30 second
Amplification- 72℃ for 30 second
Stage 3
Last Amplification- 72℃ for 5 minutes
Infinite Hold- 4℃
Aequorin Product- 295 bp
PCR Elements-
Total Reaction- 34
Total Reaction Volume- 10uL
Transgenic Plant sample- 41- 73 & 12, Aequorin Plasmid- 1

PCR Standar Total Working

Components d Reaction Volume(uL)
volume
(uL)
GT master mix 5 34 170
10uM Primer F 0.25 34 3
10uM Primer R 0.25 34 3.25
Genomic DNA 1 34 13
MQ water 3.5 34 45

13
RESULT- All samples were NEGATIVE, Positive control amplified.
Inference- NO plants carry Aequorin plasmid.

RESULT- All samples were NEGATIVE, Positive control amplified.

Inference- NO plants carry Aequorin plasmid.

14
AIM- Screening of chickpea Aequorin lines by NPTII PCR.
PCR Stages-
Stage 1
Primary Denaturation- 98℃ for 30 second
Stage 2 (35 Rounds)
Secondary Denaturation- 98℃ for 10 second
Annealing- 55℃ for 30 second
Amplification- 72℃ for 1 minute
Stage 3
Last Amplification- 72℃ for 5 minutes
Infinite Hold- 4℃
Kanamycin Product- 786 bp
PCR Elements-
Total Reaction- 10
Total Reaction Volume- 10uL
Transgenic Plant sample- 1- 8, Aequorin Plasmid- 1, Wild Type- 1
Primer used- NPTII (Kanamycin)

PCR Standard Total Working

Components volume Reaction Volume(uL)
(uL)
GT master mix 5 10 50
10uM Primer F 0.25 10 2.5
10uM Primer R 0.25 10 2.5
Genomic DNA 1 10 10
MQ water 3.5 10 35

15
RESULT- Strong band observed in sample 3 & 6, Weak band is observed in sample 1,2 & 5 and
No band in Wild type.
Inference- Plant 3 & 6 carry Aequorin plasmid and plant 1,2 & 5 shown weak band due to
chimeric effect.

16
AIM- Screening of chickpea Aequorin lines by NPTII PCR.
PCR Stages-
Stage 1
Primary Denaturation- 98℃ for 30 second
Stage 2 (35 Rounds)
Secondary Denaturation- 98℃ for 10 second
Annealing- 55℃ for 30 second
Amplification- 72℃ for 30 second
Stage 3
Last Amplification- 72℃ for 5 minutes
Infinite Hold- 4℃
Kanamycin Product- 786 bp
PCR Elements-
Total Reaction- 10
Total Reaction Volume- 10uL
Transgenic Plant sample- 8- 23, Aequorin Plasmid- 1
Primer used- NPTII (Kanamycin)

PCR Standard Total Working

Components volume Reaction Volume(uL)
(uL)
GT master mix 5 16 80
10uM Primer F 0.25 16 4
10uM Primer R 0.25 16 4
Genomic DNA 1 16 16
MQ water 3.5 16 56

17
18
RESULT- Weak band observed in sample 17.
Inference- Plant 17 shown weak band due to chimeric effect.

19
 CONCLUSION
In conclusion, sample 3 & 6 of chickpea (T0 generation) showed positive results to Aequorin
plasmid and sample 1,2,5 & 17 showed weak band due to chimeric effect in chickpea. To
confirm Aequorin gene in T1 transgenics, T1 seeds were collected from T0 plants. In next
generation, T1 transgenic plants will be screened for Aequorin gene to get stable transgenic
plants.

20