REPORT ON WORK DONE AT CENTRE FOR BIOTECHNOLOGY

ANNA UNIVERSITY, CHENNAI – 25.

Title of work: Cloning of Noda virus RNA Dependent RNA polymerase

(RDRP) in pUC1 8 vector
Outline of work:

 Total RNA Extraction

 cDNA synthesis at different concentration of RNA
 Gradient PCR- different temperature (55°C -62°C) conditions
 PCR Reaction with Taq and PR polymerase
 Large scale PCR amplification for gel elution.
 Restriction Digestion of the gene (RDRP) and pUC18 vector
 Ligation
 Ligation PCR
 Competent cell preparation and transformation
 Patch and Lysate PCR
 Plasmid Extraction
 Restriction Digestion

Total RNA Extraction

Procedure:

1. Pellet the cells (cells grown in suspension) by centrifugation and lyse the cells in
TRIZOL reagent by repetitive pipetting. Incubate the homogenized samples for 5 min at
15-30C to permit the complete dissociation of nucleoprotein complexes.

2. Add 0.2 ml of chloroform per 1 ml of TRIZOL Reagent. Cap sample tubes securely.
Shake tubes vigorously by hand for 15 seconds and incubate them at 15 -30C for 2-3
min. Centrifuge the samples at 12,000 x g for 15 minutes at 2 to 8C.

3. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5
ml of isopropyl alcohol per 1 ml TRIZOL Reagent used for the initial homogenization.
Incubate samples at 15 -30C for 10 min and centrifuge at 12,000 x g for 10 min.

4. Remove the supernatant. Wash the RNA pellet twice with 75% ethanol, adding at least 1
ml of 75% ethanol per 1 ml of TRIZOL Reagent used for the initial homogenization. Mix
the sample by vortexing and centrifuge at 7,500 x g for 5 min at 2 to 8C.

5. Briefly dry the RNA pellet. Dissolve RNA in RNase-free water, incubating for 10 min at
55-60C.
cDNA synthesis at different concentration of RNA

Procedure:

1) The isolated RNA was added with 25pmol of reverse GSP with DEPC treated water and kept
for pre-incubation at 65C for 5minutes.
2) To that add cDNA synthesis mix (5X reaction buffer, M-MuLV Reverse Transcriptase (20 U /
µl), RiboLockRNase inhibitor (20 U / µl), 10mMdNTP mix) to make it up to a Final volume of
20µl and incubate at 37C for 60 min
3) Stop the reaction by incubating for 5 min at 70C.
4) Prior to PCR, synthesized new cDNA was resolved in 0.8% Agarose gel to confirm
the purity of cDNA.
5) PCR reaction of the synthesized cDNA was carried out.
Agarose Run of total RNA and cDNA

1 2 3 4 5

Lanes: 1-total RNA, 2-cDNA(1µl), 3-cDNA(2µl), 4-

cDNA(3µl), 5- Marker 1kb

PCR at different conditions

Reaction setups:

Various PCR setups are carried out at different annealing temperature in order to get the
amplification of the specific gene.

1) Initial denaturation 94ºC for 4min

2) Denaturation 94°C for 1min
3) Annealing 65C for 1min
4) Extension 72ºC for 4min
5) Final extension 72°C for 5min for 30 Cycle Using Taq DNA polymerase

The gene was amplified with multiple bands and the PCR product failed to reamplify.
 In order to trouble shoot, gradient PCR was run at different annealing temperature from
55 C to 620C with reaction setup of
0

1) Initial denaturation 950C for 5min

2) Denaturation 950C for 1min
3) Annealing 550C-620C for 40sec
4) Extension 720C for 3.5min
5) Final extension 720C for 10min for 35Cycle Using Taq DNA polymerase.

The amplification of the gene was achieved at 55, 59, 61, 62ºC, but the intensity of the band after
amplification was faint.

In order to get a good amplification the reaction setup was done with PR polymerase
enzyme for both the cDNA and PCR product with same reaction setup with annealing
temperature 590Cand 62°C, we got amplification at 3.1kb but with multiple bands.

Gradient PCR at different annealing temp (55-62) PCR with PR polymerase

3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6
Lane 3-1kb ladder, 4-NC, 5-62C, 6-61C, 7-60C, 8- Lane 1 and 2 cDNA, 3and4- PCR product,
59C, 9-58C, 10-57C,11-56C,12-55C 5-NC, 6- 1kb ladder

PCR Reaction with Taq and PR polymerase.

The PCR reaction was carried out with both Taq and PR polymerase at annealing
temperature 64°C for 30sec and extension 72ºC for 3min. We got good amplification of the gene
with Taq polymerase.

The cDNA was prepared freshly and PCR was run with Taq and good amplification was
observed.
 3.1kb

1 2 3 4 5

Lane1-cDNA+Taq,2-same,3-cDNA with PR, 4-

same, 5- ladder 1kb

Large scale PCR amplification for gel elution:

The cDNA was amplified in large scale with Taq at annealing temp 640C for 30sec and
gel was eluted with RBC kit and the eluted DNA was quantified using Nanodrop before
restriction digestion.

1 2

Lane-1 Eluted DNA, 2-1kb ladder

Restriction Digestion of the gene (RDRP)

Gene 3µg(55µl)
Buffer 10µl(10x)(NEB 4)
Enzyme EcoR1 and Sal1 1.5+1.5µl
Water 30µl
Restriction Digestion of the Vector (pUC 18)

Gene 2µg (60µl)

Buffer 10µl s(10x)
Enzyme EcoR1 and Sal1 1.5+1.5µl
Water 27µl
The above mentioned cocktail of Restriction digestion were kept for incubation at 37ºC for 3
hours and the digested product was checked in 0.8% Agarose gel and eluted using RBC gel
elution kit and the eluted product was also checked in 0.8% Agarose gel and quantified using
nanodrop and was found to be 11.6ng/ µl (gene) and 6.2ng/µl(vector)

1 2 3

Lane1-Vector pUC18, 2-1 kb ladder, 3-RDRP

gene(3.1k)

Ligation

T4 ligase Enzyme 1µl

Buffer 2 µl
Gene (RDRP) 9 µl (11.6 ng/µl)
Vector (pUC18) 5 µl (6.2ng/µl)
 Water 3 µl
Incubate at 22°C for 1 hour and 16ºC overnight.
Ligation PCR:

The PCR reaction were carried out using ligated product as template using T7 forward
primer and gene specific reverse with a control of cDNA with GSP. There was no amplification
in the ligated product.

1 2 3

Lane1- ligation PCR,2-cDNA,3-1kb ladder

Competent cell preparation

1) Take DH5α culture at OD 0.6 and kept in ice for 30min

2) Spin the culture at 4000rpm for 10 min at 4°C
3) Discard the supernatant and to the pellet add 1/4th volume of 0.1M cacl2
4) Keep the suspension in ice for 15minutes
5) Centrifuge the cells at 4000rpm for 10minutes at 4ºC
6) Discard the supernatant to the pellet add ½ the volume of cacl2
7) Keep the suspension in ice for 30minutes
8) Spin the culture at 4000rpm for 10minutes at 4°C
9) Discard the supernatant and to the pellet add 1-2 ml of cacl2

Transformation

1) 5µl of ligation mixture was added to the 100µl of the prepared competent cells.
2) Keep in ice for 30minutes
3) Heat shock at 42C for 90seconds
4) Snap chill in ice for 5 minutes
5) To that add 400µ of LB medium and incubate at 37C for 45mim-1hour at shaker
6) Spin at 400rpm for 5minutes
 7) Remove 300-400µl of the supernatant and plate the remaining cells in LB Amp plate and
kept at incubation at 37ºC.

Patch and Lysate PCR

1) 5 colonies were transformed and patch work was done in the fresh LB Amp plate and
kept at incubation at 37°C
2) The grown culture were inoculated in 50µl of distilled water and kept at 100°C for
10minutes
3) Immediately chill it on ice for 5minutes and spin it at 8000rpm for 10minutes
4) The supernatant was taken as template and PCR was done with GSP
5) All the five colonies gave good amplification at 3.1kb which was checked in 0.8%
Agarose gel.

1 2 3 4 5 6
Lane 1-5 Clones 1-5, 6- 1kb ladder
Plasmid Extraction

1) The colonies were inoculated in LB broth and kept at incubation at 37ºC

2) Then the cultures were centrifuged and the supernatant was discarded.
3) The pellet was used for the plasmid isolation using Qiagen plasmid isolation kit as
per the manufactures instruction.
6) The extracted plasmid was checked using 0.8% Agarose gel and the plasmid was
quantified using nanodrop.

Restriction Digestion

The extracted plasmids were restricted digested using EcoR1 and Sal1 enzyme and the
presence of the insert was checked using 0.8% Agarose gel.

Reaction Condition:
Plasmid 2µl(511ng/µl)
Enzyme (EcoR1&Sal1) 1µl each
NEB buffer 2µl
Water 14µl
Incubated at 37ºC for 4 hours.

Insert 3.1kb

3kb
Vector 2.8kb

1 2 3 4
Lane 2-1kb ladder 3- uncut plasmid,
4and5-digested plasmid