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Presented by- Ravi Ranjan
Experiments
performed:-
DNA isolation from Jatropha curcas plant sample
RNA isolation form Jatropha curcas plant sample
De...
Jatropha curcas
Jatropha is poisonous, semi evergreen shrub
(6m tall).
They are also grown in deserts.
Seeds of jatropha a...
DNA ISOLATION BY
C TAB METHOD
DNA is a higher molecular weight
molecule and can be isolated from
plant tissue by fracturin...
C TAB method of DNA isolation
Leaf sample is taken, grinded, and taken in micro-centrifuge tube,
pre-warmed C TAB is added...
RNA ISOLATION BY
C TAB METHOD
RNA can be isolated from plant
tissue by C TAB method.
The isolation of RNA is
preferred ove...
C TAB method of RNA isolation
Leaf sample is taken, grinded, and taken in micro-centrifuge tube, extraction
buffer is adde...
Determination of quality and quantity of
isolated
DNA and RNA
The quality and quantity of the genomic
DNA/RNA was checked ...
Casting of Agarose gel
 The casting unit was setup.
 0.8% Agarose DNA, and 1.2% RNA Agarose is dissolved in
100ml of 1x ...
DNA and RNA bands observed in UV Transilluminator
Polymerase chain reaction (PCR)
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant s...
1.
• Denaturation
2.
• Annealing
3.
• Extension
3.
Primary Denaturation
Final Extension
X 35
Cloning
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of q...
Isolation of plasmid DNA
(from E. coli)
The cells are centrifuged and, resuspended pellet in different
buffers(STE buffer,...
Ligation
Reagents Volume used
5X ligation buffer 2µl
PGEM-T vector 1µl
T4 ligase 1µl
Sterile water Add to final volume of ...
Competent cell preparation
Single colony is picked up and grown overnight.
Pre-warmed algae media is added and stored at 3...
Transformation
competent cells and DNA is added in eppendroff tubes, stored in ice for 3o
minutes, and then transferred to...
Screening
+ Amp
Recombinant plasmid isolation
white colonies are picked and inoculated in 3 ml LB broth and incubated
at 37 ˚C for overnig...
molecular biology techniques -jaypee university of information technology- ravi ranjan
molecular biology techniques -jaypee university of information technology- ravi ranjan
molecular biology techniques -jaypee university of information technology- ravi ranjan
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molecular biology techniques -jaypee university of information technology- ravi ranjan

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

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molecular biology techniques -jaypee university of information technology- ravi ranjan

  1. 1. Presented by- Ravi Ranjan
  2. 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. 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. 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. 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. 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. 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. 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. 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.
  10. 10. DNA and RNA bands observed in UV Transilluminator
  11. 11. 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.
  12. 12. 1. • Denaturation 2. • Annealing 3. • Extension 3. Primary Denaturation Final Extension X 35
  13. 13. 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
  14. 14. 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.
  15. 15. 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.
  16. 16. 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.
  17. 17. 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.
  18. 18. Screening + Amp
  19. 19. 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.

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