JIB322 / 3 JIB322LR1 BIOLOGI MOLEKUL MOLECULAR BIOLOGY Academic Session 2017/2018 LAB REPORT LR1 Name
MUHAMMAD NURMAN BIN SAHAID
Matric No.
JP/9571/15
IC No.
841009145879
Re Regional Centre
KUALA LUMPUR
Phone No.
House : H/P: 013-2363484
841009145879
JIB 322 PRACTICAL
DATE OF PRACTICAL: 22 Januari 2018 - Sesi 4pm - 7pm 28 Januari 2018 - Sesi 8pm - 11pm 29 Januari 2018 - Sesi 12pm - 3pm 30 Januari 2018 - Sesi 12pm - 3pm
GROUP 4 NAMES OF GROUP MEMBER:
MUHAMMAD NURMAN BIN SAHAID ( JP/9571/15 ) MOHD HANIF BIN MOHD KASMURI ( JP/9149/14 ) MOHD TARMIZI BIN CHE ALI ( JP/9544/15 ) MOHD SHAHRIMAN BIN GHAZALI ( JP/9162/14 ) MOHD NAZRI SHAIDIN ( JP/9157/14 )
JIB 322 PRACTICAL
DATE OF PRACTICAL: 22 Januari 2018 - Sesi 4pm - 7pm 28 Januari 2018 - Sesi 8pm - 11pm 29 Januari 2018 - Sesi 12pm - 3pm 30 Januari 2018 - Sesi 12pm - 3pm
GROUP 4 NAMES OF GROUP MEMBER:
MUHAMMAD NURMAN BIN SAHAID ( JP/9571/15 ) MOHD HANIF BIN MOHD KASMURI ( JP/9149/14 ) MOHD TARMIZI BIN CHE ALI ( JP/9544/15 ) MOHD SHAHRIMAN BIN GHAZALI ( JP/9162/14 ) MOHD NAZRI SHAIDIN ( JP/9157/14 )
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JP/ 9571/ 15 PRACTICAL 1
TITTLE: The Use of Restriction Enzyme for Digestion of DNA ABSTRACT: In this experiment, the plasmid –with and –without insert were digested by an appropriate restriction endonuclease. The sequence of original plasmid named pEF1a, Pef1A-3D5 and pNL4-3 was obtained from e-learning usmpjj portal website and was exported into Benchling
software for virtual analysis. The restriction enzyme was used in this experiment was BsaWI which
obtaining
from
NEB
company.
at the optimal temperature
60˚C for
The
enzyme
cut
at
restriction
site
1 hours. The digestion of 200 ng plasmid -
without insert resulted in 2 bands. The patterns of digestion were different between plasmid – with and –without insert.
INTRODUCTION 1. DNA plasmid Plasmids are small, circular duplex DNA molecules (Fursule et all, 2006). all, 2006). A particularly useful plasmid vector for selecting transfected cells called pBR322 is itself a hybrid plasmid. This plasmid contains two genes, amp and tet, which confer resistance to penicillin and tetracycline,respectively. (Geoffrey, 1998). 2. Restriction Enzyme
Systemic cleavage of duplex DNA at specific sites require restriction enzymes. Most of these enzymes recognize a sequence of either four or six contiguous base pairs. The cleavage sites are situated so that blunt-ended or staggered-ended DNA results from the cleavage reaction. (Geoffrey, 1998).
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3. Benchling software
Benchling, Inc. develops an integrated software solution for experiment design, note-taking, and molecular biology. The company offers Benchling, a research platform that allows scientists to design, share, and record experiments on one interface; and Bioregistry system, a biological entity registration and strain management solution. Its platform consists of an integrated lab notebook; biological registration system to track inventory, such as plasmids, antibodies, and cell strains; and molecular biology design and analysis suite that streamlines research and development from start to finish. (https://www.bloomberg.com) OBJECTIVE: To digest 3 DNA plasmids named pEF1a, Pef1A-3D5 and pNL4-3 by using a restriction enzyme called BsaWI. MATERIALS AND METHODS: 1.
Reagent and equipment used Three microtubes containing DNA plasmid sample with each contains 10uL of 40ng/uL pEF1a, 10uL of 40ng/uL pEF1a-3D5 and 10uL of 40ng/uL pNL4-3. Another microtube containing 14uL of 10X cut smart and 20uL of 1U/5uL restriction enzyme (BsaWI). One centrifuge tube containing 76uL of distilled water. 8 clean microtubes and rack are provided for test with box containing ice cubes. Micropipette 0.5-10uL with pipette tips. Other equipment that are provided includes electric mixer, microtube centrifuge, Bio-Rad PCR Thermal Cyclers and a -20oC freezer. 7 clean microtubes are labelled as 1a (cut pEF1a), 1b (uncut pEF1a), 2a (cut pEF1a-3D5), 2b (uncut pEF1a-3D5), 3a (cut pNL4-3), 3b (uncut pNL4-3) and C (control). The remaining 1 clean microtubes will be used as balancing in the centrifuge machine. For microtube 1a, 5uL of 40ng/uL pEF1a are transferred, then adding 2uL of 10X cut smart, 8uL of distilled water and 5uL of 1U/5uL restriction enzyme (BsaWI) making the total solution is 20uL with DNA concentration of 200ng. For microtube 1b, 5uL of 40ng/uL pEF1a are transferred, then adding 2uL of 10X cut smart, and 13uL of distilled water making the total solution is 20uL with DNA concentration of 200ng. For microtube 2a, 5uL of 40ng/uL pEF1a-3D5 are transferred, then adding 2uL of 10X cut smart, 8uL of distilled water and 5uL of 1U/5uL restriction enzyme (BsaWI) making the total solution is 20uL with DNA concentration of 200ng. For microtube 2b, 5uL of 40ng/uL pEF1a -3D5 are transferred, then adding 2uL of 10X cut smart, and 13uL of distilled water making the total solution is 20uL with DNA concentration of 200ng. For microtube 3a, 5uL of 40ng/uL pNL4-3 are transferred, then adding 2uL of 10X cut smart,
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8uL of distilled water and 5uL of 1U/5uL restriction enzyme (BsaWI) making the total solution is 20uL with DNA concentration of 200ng. For microtube 3b, 5uL of 40ng/uL pNL4-3 are transferred, then adding 2uL of 10X cut smart, and 13uL of distilled water making the total solution is 20uL with DNA concentration of 200ng. In the microtube C, 2uL of 10X cut smart is transferred and added by 13uL of distilled water and 5uL of 1U/5uL restriction enzyme (BsaWI) making the final solution is 20uL. All the microtubeare mixed thoroughly with electric mixer and spin for a few second using a centrifuge to make sure the solution is at the bottom of the microtube. All the microtube is placed in a box containing ice while waiting for the Bio-Rad PCR Thermal Cyclers. After placing all the 5 group microtubes into the Bio-Rad PCR Thermal Cyclers, it was set for 60oC for 1 hour. After the incubation is completed, the microtube is kept in -20 oC freezer for Practical 2. 2. The flow chart of method
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3. Table of reaction (calculation) DNA plasmid concentration in each microtube (1a, 1b, 2a, 2b, 3a and 3b) Volume in each microtube with BsaWI (cut) (1a, 2a and 3a) 5uL DNA plasmid, 2uL 10X cut smart, 8uL H20, 5uL of BsaWI Volume in each microtube without BsaWI (uncut) (1b, 2b and 3b) 5uL DNA plasmid, 2uL 10X cut smart, 13uL H20 Volume in microtube C (control) 2uL 10X cut smart, 13uL H20, 5uL of BsaWI
40ng/uL x 5uL = 200ng 5 + 2 + 8 + 5 = 20uL
5 + 2 + 13 = 20uL
2 + 13 + 5 = 20uL
RESULT: 1. In-silico results (full-length plasmid size, restriction sites for enzyme) Plasmid name Size (kb) Restriction site for BsaWI No. of RE site found pEF1a
7.056
6
pEF1a-3D5
7.593
7
pNL4-3
9.709
3
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2. Virtual digest image from Benchling software
Figure 1 shows image from Benchling software that shows the virtual digest of 3 different DNA plasmid named pEF1a, pEF1a-3D5 and pNL4-3 with and without restriction enzyme called BsaWI. Legends: (1) pEF1a with BsaWI, (2) pEF1a without BsaWI, (3) pEF1a-3D5 with BsaWi, (4) pEF1a-3D5 without BsaWI,(5) pNL4-3 with BsaWI and (6) pNL4-3 without BsaWI
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4. Result description: The band at the left side of the virtual digest is known as Ladder which range from 100bp to 12.0kb and act as a ruler for the test band. There are 3 plasmids used in this experiment which is pEF1a, pEF1a-3D5 and pNL4-3 with a restriction enzyme (RE) called BsaWI. The BsaWI identified the restriction site which consist of
. Number 1 is pEF1a with BsaWI showing 6 bands, which
means the RE cuts at 6 site, the bands also shows the size of each cut DNA is 3.975kb, 1.649kb, 831bp, 379bp, 147bp and 75bp (but because of the ladder start at 100bp the 75bp band is at 100bp). Number 2 is pEF1a without BsaWI means no cutting and the band shows the full length of the DNA plasmid which is 7.056kb. Number 3 is pEF1a-3D5 with BsaWi showing there is 7 bands which means the RE cuts at 7 site with each size of cut DNA is 2.633kb, 1.879kb, 1.649kb, 831bp, 379bp, 147bp and 75bp (again the band show at 100bp). From the band we can see that number 1 and number 3 have 5 bands that are similar from 100bp to 1.649kb. Number 4 is pEF1a-3D5 without BsaWI means uncut DNA plasmid showing the full size of the DNA marked by only 1 band at 7.593kb. Number 5 is pNL4-3 with BsaWI showing there is 3 bands which means the RE cuts at 3 site with each size of cut DNA is 5.898kb, 3.177kb and 634bp. Number 6 is pNL4-3 without BsaWI means uncut DNA plasmid will show full size of the DNA marked by only 1 band at 9.709kb
DISCUSSION 1. State the type of DNA markers (ladders) and their functions.
DNA markers, to identify target band ranging over a length of 20000 nucleotides.
Protein markers, for the analysis of proteins cleaved at different site.
RNA markers, to identify RNA fragment within a length of dozen nucleotides.
2. State the controls and describe their importance in this experiment. The controls used are reagent blank control shown by no band forming on the gel. It consist of all reagents (10X cut smart, H 2O and BsaWI) used during sample processing but contains no DNA plasmid sample. This control is used to detect DNA contamination of the analytical reagents used to prepare the sample for analysis.
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3. Briefly explain why two confirmations of plasmid (cut and un-cut plasmids) used in this experiment? Un-cut plasmids are used as a control to ensure that the restriction enzymes are working. When this uncut DNA plasmid is isolated and run on an agarose gel, there would be 3 bands form which is supercoiled, relaxed and nicked. If the cut DNA plasmid looks like the un-cut DNA plasmid then the enzymes are not working. 4. State the form of DNA end produced when restricted with the above enzyme. BsaWI produce a blunt end on the DNA end when the restriction ezyme is used. 5. Give one application of restriction enzyme. Restriction enzyme can be used in genetic engineering because they can cut fragments of DNA that contain genes. 6. Scenario You are given with full-length plasmid pUC19 from Addgene (size : 2.686kb) to be restricted with NotI enzyme. The restriction sequence for NotI enzyme is (
). Upon analysis
with Benchling web application, you found there (was no single ) restriction site/s throughout the plasmid. Based on the scenario above, show the analysis and predict the outcome of this experiment.
Because there is no single restriction site throughout the plasmid, the number of cut is 0 which means the plasmid is uncut.
On the gel electrophoresis, the single band shows the full size of the plasmid which is 2686bp.
If there is a new gene added to this plasmid there will be no gene recombination on this uncut DNA plasmid.
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CONCLUSION: Restriction enzyme can be used to cut DNA plasmid if there is restriction site found throughout the plasmid. The number of the restriction site found will be the number of cut for the restriction enzymes, while the band produce showed the length or size for plasmid.
REFERENCES: 1. Gerald, K., Cell and Molecular Biology: Concepts and Experiments, 2nd Edition, New York, John Wiley & Sons. 1996. 2. Geoffrey, Z., Biochemistry 4th Edition, United States of America, Wm. C. Brown Publishers. 1998. 3. William, D., S., Jamie, S., C., Raul, L., C., Schaum’s Easy Outlines: Molecular and Cell Biology, United States of America, Mc Graw Hill. 2003. 4. Fursule, R., A., Kulkarni, J., S., Agarkar, P., H., Biochemistry: Basic and Applied, India, Nirali Prakashan. 2006. 5. Aniruddha, D., Edward, R., D., Introdction to Genomic Signal Processing with Control, Florida, CRC Taylor & Francis Group. 2007. 6. https://www.bloomberg.com/research/stocks/private/snapshot.asp?privcapid=261131503 7. https://benchling.com
JIB322 / 3 JIB322LR2 BIOLOGI MOLEKUL MOLECULAR BIOLOGY Academic Session 2017/2018 LAB REPORT LR2 Name
MUHAMMAD NURMAN BIN SAHAID
Matric No.
JP/9571/15
IC No.
841009145879
Regional Centre
KUALA LUMPUR
Phone No.
House : H/P:013-2463484
841009145879
841009-14-5879
JP/ 9571/ 15 PRACTICAL 2
TITTLE: Southern Blotting for Identified Plasmid DNA Sequence ABSTRACT In this Southern blotting experiment, the restricted products obtained from practical 1 were separated according to size by gel electrophoresis through a standard 0.8% agarose gel. The DNA was then denatured in situ and transferred from the gel to a nitrocellulose membrane. The position of the DNA fragments are at the same time during the transfer process. INTRODUCTION
1. Gel electrophoresis Gel electrophoresis can be used to separate DNA fragments on the basis of their sizes. (Aniruddha et all,2007). It involves the movement of charged molecules or ions through semisolid support medium under the influence of an electrical field. Agarose gel, the common media for this procedure will cast as a thin slab in a mold with sample well at one end, is submerged in a buffer solution with the sample well side toward negative pole (cathode). (William et all , 2003). 2. Southern Blotting It involves restriction enzyme digestion and agarose gel electrophoresis of the target DNA prior to the hybridization assay. The different bands on the agarose gel are transferred by capillary action onto a nitrocellulose or nylon membrane in a blotting apparatus. During the transfer, each of the DNA bands is transferred onto the membrane in the same relative position that it had in the gel.(William et all ,2003). 3. DNA denaturation Polymerase Chain Reaction (PCR) mixture is heated at 95°C for 15-30 seconds. The heat separates double stranded DNA into single strand which can act as templates for new DNA synthesis. (Fursule et all, 2006). The two strands of the target DNA molecules are separated by the heating the DNA to break the hydrogen bonds between bases, yielding two separate strand. (William et all, 2003)
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OBJECTIVE: To transfer restricted DNA plasmid from gel to a nitrocellulose membrane through the Southern Blotting MATERIALS AND METHODS 1. Reagent and equipment used. All samples from practical 1 are taken out from the freeze for gel electrophoresis. The gel electrophoresis are being set up with a 0.8% agarose gel that has been prepared previously and 1 L of TBE buffer are pour inside to flooded the gel chamber. The gel are handle with care because ethidium bromide is a mutagen and carcinogenic. Remove the combs from the gel. The 1st well on the gel is set for a 1kb ladder. Pipette 0.5uL of 1kb ladder (500ng/uL) and mixed it with 1uL of dye and 4.5uL of buffer. This will produce a 6uL 250ng concentration of 1kb ladder which will be loaded carefully into the 1st well. Pipette 5uL of sample 1b (uncut pEF1a), add 1uL of dye, mixed and load into the 2nd well. Pipette 5uL of sample 1a (cut pEF1a), add 1uL of dye, mixed and load into the 3rd well. Pipette 5uL of sample 2b (uncut pEF1a-3D5), add 1uL of dye, mixed and load into the 4th well. Pipette 5uL of sample 2a (cut pEF1a-3D5), add 1uL of dye, mixed and load into the 5th well. Pipette 5uL of sample 3b (uncut pNL4-3), add 1uL of dye, mixed and load into the 6th well. Pipette 5uL of sample 3a (cut pNL4-3), add 1uL of dye, mixed and load it into the 7th well. Pipette 5uL of sample C (control), add 1uL of dye, mixed and load it into the 8th well. Pipette 5uL of sample 3D5 (positive control), add 1uL of dye, mixed and load it into the 9th well. The 10th well is set for another 1kb ladder. Pipette 0.5uL of 1kb ladder (500ng/uL) and mixed it with 1uL of dye and 4.5uL of buffer. This will produce a 6uL 250ng concentration of 1kb ladder which will be loaded carefully into the last well. Close the lid of the electrophoresis chamber and connect all the negative and positive electrodes. Set the current at 110V for 40 minutes. The process is start when tiny
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bubbles seen in the chamber. After the time is stop, shut the power off and remove the gel from the chamber for gel documentation. The gel is trimmed at 83x83cm and place into a plastic container to prepare the DNA for transfer. Add 0.25M HCl into the gel inside the plastic container for 15 min and shakes the plastic container. Rinse the gel with distilled water. Add 1.5M NaCl, 0.5M NaOH into the gel inside the plastic container for 15 min and shakes the plastic container. Repeat with the same 1.5M NaCl, 0.5M NaOH for another 15 min. Rinse the gel with distilled water. Add 1.5M NaCl, 0.5M Tris-Cl pH 7.0 into the gel inside the plastic container for 15 min and shakes the plastic container. Repeat with the same 1.5M NaCl, 0.5M Tris-Cl pH 7 yn b7bn utym b
.0 for another 15 min.
Wet the nitrocellulose membrane with 20X SSC and do not remove solution. The southern blot arrangement is prepared according the diagram provided. The surface of the gel with band is making sure to facing the wet nitrocellulose membrane. The southern blot is let to run for about 12 to 14 hours. 2. The flow chart of method
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3. Diagram/image of southern blotting transfer apparatus
RESULT
Figure 2 shows the bands of pEF1a, Pef1A-3D5 and pNL4-3 plasmid restricted by restriction enzyme called BsaWI
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3. Result description Based on the result, the band on the left and right side is the 1kb ladder that act like a ruler for the other bands. There are three DNA plasmid provided in this practical which is pEF1a, pEF1a3D5 and pNL4-3. The restriction enzyme use is BsaWI. The 1b is the pEF1a without BsaWI or uncut showing 2 bands at which the first band is higher than the second band. This band show the full size of the uncut plasmid, while the more than one band is cause by the DNA supercoiled, nicked or relaxed. The 1a is the pEF1a restricted by BsaWI showing 4 bands, the bands are much lower than the 1b shows that the restricted plasmids DNA have less size compare to uncut. The 2b is the pEF1a-3D5 without BsaWI or uncut showing 2 bands at which the first band is higher than the second band. This band shows the full size of the uncut plasmid, while the more than one band is caused by the DNA supercoiled, nicked or relaxed. The 2a is the 841009-14-5879
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pEF1a-3D5 restricted by BsaWI showing 4 bands, the bands are much lower than the 2b shows that the restricted plasmids DNA have less size compare to uncut. We also can see that the band between 1a (cut pEF1a) and 2a (cut pEF1a-3D5) has a similar pattern of 2nd to 4th bands. This shows that the second, third and fourth fragment of restricted plasmids have the same size. The 3b and 3a have a long line that stop at the same lowest band which is undetermined probably that the molecules of pNL4-3 is smaller and move at a greater speed. The C is a control blank where no bands is formed due to no DNA is added. The 3D5 is a positive control without any restriction enzyme, showing 2 bands at which the first band is higher than the second band. This band show the full size of the uncut plasmid, while the more than one band is cause by the DNA supercoiled, nicked or relaxed. DISCUSSION 1. Explain why low percentage of agarose (0.8%) used in this experiment? Low percentage of agarose (0.8%) is suitable because it allowed optimum resolution for linear DNA (about 800-12000 bp). Higher percentage of agarose will cause the ladder
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band formed very near each other or cluttered thus make it difficult to see the resolution for linear DNA 2. What will happen if 10X TBE is used in electrophoresis buffer and explain? 10X TBE cannot be used. In pure concentration the TBE will not working in the experiment. It must be diluted to a working solution of 1X TBE. 3. State the principle of gel electrophoresis and explain. Gel electrophoresis seperates DNA fragments according to molecular size. The DNA is placed in the well after the combs is removed, the negatively charged DNA molecule will move down the gel to the positive electrode. Smaller DNA molecules can travel much further through the gel small pores compare to larger DNA molecule. The size of the molecules is shown by the bands on the gel. 4. Why low signal intensity seen in low molecular weight DNA? Low molecular weight DNA moves very fastest through the gel pores, thus lessening the intensity of its signal. 5. Explain why the plasmid -with insert has more restricted bands compared with the plasmid -without insert? The restriction enzyme in the plasmid –with insert can recognise the restriction site thus cutting the DNA. The more restriction site is recognise, the more restricted the plasmid DNA. These restricted DNA have different size that will be shown by the band on the gel, thus making more bands forming than the plasmid –without insert. The plasmid – without insert still has its circular pattern and is more complex, twisted and knotted and will produce a less band. 6. State the principle of southern blotting and explain Southern blotting is based on the principles of separation of DNA fragments by gel electrophoresis, transferred to a nitrocellulose membrane and identified by labelled probe hybridisation. After DNA fragments is separate by gel electrophoresis it is transfer by the
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means of southern blotting to a nitrocellulose membrane. The DNA is hybridising with a probe that is labelled and the probe will identified complimentary target DNA. 7. Scenario From the Benchling, the highest band is below 1kb. This means the restricted DNA has smaller size molecules. The percentage of gel that is suitable for used is 0.8% and the marker type suitable for used is 100bp for a proper ladder for small restricted DNA molecules. Percentage of agarose gel need = 0.8% Volume of agarose gel need, Weight
of
agarose
= 100 ml
need,
=
?g,
Based
on
0.8
%
(
/ ),
Preparation: weight 0.8 g of agarose gel and add 100 ml of 0.5X TBE, heat and let solidify. CONCLUSION: The DNA fragment from gel electrophoresis is transferred from the agarose gel onto nitrocellulose membrane. The bands on the gel can be seen in the result (Figure 2). REFERENCES: 1. Gerald, K., Cell and Molecular Biology: Concepts and Experiments, 2nd Edition, New York, John Wiley & Sons. 1996. 2. Geoffrey, Z., Biochemistry 4th Edition, United States of America, Wm. C. Brown Publishers. 1998. 3. William, D., S., Jamie, S., C., Raul, L., C., Schaum’s Easy Outlines: Molecular and Cell Biology, United States of America, Mc Graw Hill. 2003. 4. Fursule, R., A., Kulkarni, J., S., Agarkar, P., H., Biochemistry: Basic and Applied, India, Nirali Prakashan. 2006.
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5. Aniruddha, D., Edward, R., D., Introdction to Genomic Signal Processing with Control, Florida, CRC Taylor & Francis Group. 2007. 6. https://www.bloomberg.com/research/stocks/private/snapshot.asp?privcapid=261131503 7. https://benchling.com
JIB322 / 3 JIB322LR3 BIOLOGI MOLEKUL MOLECULAR BIOLOGY Academic Session 2017/2018 LAB REPORT LR3 Name
MUHAMMAD NURMAN BIN SAHAID
Matric No.
JP/9571/15
IC No.
841009145879
Regional Centre
KUALA LUMPUR
Phone No.
House : H/P:013-2463484
841009145879
841009-14-5879
JP/ 9571/ 15 PRACTICAL 3
TITTLE: Southern hybridization of Digixonin-labeled probes to DNA immobilized on nitrocellulose membrane ABSTRACT In this experiment, the nitrocellulose membrane was soaked in 20X SSC and baked in 90 C vacuum oven before proceeded with the Southern hybridization. These steps are intended to permanently attach The DNA to the membrane. Then, Southern hybridization was performed to detect a specific DNA sequence in a probe labelled of DNA fragments. The strength of the signal depends on several factors such as the proportion of immobilized DNA that is complementary to the probe, the size of the DNA molecules and its specific activity, the temperature of hybridization process and the amount of DNA transferred to the nitrocellulose membrane.
INTRODUCTION
1. DNA Fixation DNA fixation is a process to permanently attach the transferred DNA to the surface of the nitrocellulose membrane. In this experiment the membrane is baked in a vacuum oven at 80oC for 1.5 hours. Another method of fixation is through exposure to ultraviolet radiation for nylon membrane. 2. Hybridization This technique is used to detect the presence of DNA from pathogen in clinical specimens and to locate specific genes in cells. It takes advantage of the ability of the nucleic acid to form stable, double stranded molecules when two single strands with complimentary bases are brought together under favorable conditions. DNA from virus or cell is denatured with alkali to separate the strands. (William et all,2003
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3. DNA probe DNA probe is a fragment of DNA of 100-1000bp which can be radioactively labelled. Other label that is used to this DNA probe is fluorescent or chromogenic dye. DNA probe can be used in DNA samples to detect the presence of nucleotide sequences (target DNA) that are complementary to the sequence in the probe. OBJECTIVE: To hybridize the immobilize DNA fragment on the nitrocellulose membrane with a DIG-labelled DNA probe. MATERIALS AND METHODS 1. List out all the reagent and equipment used The nitrocellulose membrane is cut at one edge and mark by a marker pen to show the surface of which the DNA fragment is. The membrane is place inside a plastic container and soaked with 2X SSC. Removed the 2X SSC and place the membrane between 2 piece of Whatman 3MM paper for drying by load the membrane to vacuum oven at 90oC for 1 hour 30 minutes. Place the membrane into a plastic container and pour in 10ml of DIG Easy Hyb, closed the container lid and put in waterbath t 37oC for 30 minutes. Wet the internal surface of Hargan tube with small amount of DIG Easy Hyb to make the insertion of the membrane is easy. Roll the membrane with the inner surface facing inwards and push it into the Hargan tube. Put in 3.5uL probe and the remaining from 5ml of DIG Easy Hyb. Place the Hargan tube to the Hargan tube rotation incubator at 60oC for 12-14 hours
841009-14-5879 2. The flow chart of method:
DISCUSSION 1. Formula for Tm calculation and Topt calculation
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2. Calculate the melting temperatures and optimal temperatures for the following DNA inserts and your calculation steps: a. lacZa gene (size : 156bp) from pUC19 plasmid (size : 2.686kb) Print Screen of pUC19 plasmid map:
Calculation:
841009-14-5879 b. Ampicillin gene (size : 861bp) from pBR322 plasmid (size : 4.361kb) Print Screen of pBR322 plasmid map:
Calculation:
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841009-14-5879 c. lacI gene (size : 1.092kb) from pET-28 a (+) plasmid (size : 5.369kb) Print Screen of pET-28 (+) plasmid map:
Calculation:
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CONCLUSION: The hybridisation of the nucleic acid requires optimum temperature to allow optimal rate of activity occur. The melting point, duration and optimal temperature of every enzyme differ according to each respective base sequences in different plasmids. REFERENCES: 1. Gerald, K., Cell and Molecular Biology: Concepts and Experiments, 2nd Edition, New York, John Wiley & Sons. 1996. 2. Geoffrey, Z., Biochemistry 4th Edition, United States of America, Wm. C. Brown Publishers. 1998. 3. William, D., S., Jamie, S., C., Raul, L., C., Schaum’s Easy Outlines: Molecular and Cell Biology, United States of America, Mc Graw Hill. 2003. 4. Fursule, R., A., Kulkarni, J., S., Agarkar, P., H., Biochemistry: Basic and Applied, India, Nirali Prakashan. 2006. 5. Aniruddha, D., Edward, R., D., Introdction to Genomic Signal Processing with Control, Florida, CRC Taylor & Francis Group. 2007. 6. https://www.bloomberg.com/research/stocks/private/snapshot.asp?privcapid=261131503 7. https://benchling.com
JIB322 / 3 JIB322LR4 BIOLOGI MOLEKUL MOLECULAR BIOLOGY Academic Session 2017/2018 LAB REPORT LR4 Name
MUHAMMAD NURMAN BIN SAHAID
Matric No.
JP/9571/15
IC No.
841009145879
Regional Centre
KUALA LUMPUR
Phone No.
House : H/P:013-2463484
841009145879
841009-14-5879
JP/ 9571/ 15 PRACTICAL 4
TITTLE:Detection of Immobilized DNA By Enzymatic Reaction
ABSTRACT In this immunodetection experiment, the DIG-labeled probes bound with immobilized DNA on nitrocellulose membrane were detected by enzymatic reaction. The probe was then
bound with Anti-digoxigenin antibody conjugated alkaline phosphatase at dilution 1:5000 in Blocking solution. The colour precipitate (colour: bluish purple) by NBT/BCIP start to form within 20minutes.
INTRODUCTION 1. Immunodetection Immunodetection is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. After nonspecific binding sites are blocked, the membrane is probed with primary antibody (digoxigenin antibody) and washed. The antibody-antigen complexes are tagged with alkaline phosphatase (or horseradish peroxidase) conjugated to a secondary anti-IgG antibody (anti-digoxigenin antibody), and detected using appropriate chromogenic (or luminescent) substrates. 2. Alkaline phosphatase Alkaline phosphatase (ALP) is homodimeric protein enzyme of 86 kilodaltonns, meaning it is formed with two molecules. Three metal ions, two Zn and one Mg, are contained in catalytic sites and both types are crucial for enzymatic activity to occur. ALP funtionsbect under alkaline pH environments and has the physiological role of dephosphorylating compounds.
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3. Colorimetric substrate Colorimetric Alkaline phosphatase system use soluble BCIP (5-bromo-5-chloro-3-indolyl phosphate) and NBT (nitrobluetetrazolium) as substrates to produce a stable reaction product that will not fade. This process can easily be inactivated by exposure to acidic solutions. This substrate will produce blue colour precipitate that is visible to the naked eye. OBJECTIVE To detect the DIG-labelled probes bound with immobilized DNA on nitrocellulose membrane by enzymatic reaction that produce colour precipitate. MATERIALS AND METHODS
1. List out all the reagent and equipment used Take out the nitrocellulose membrane from the Hargan tube and place the membrane inside the plastic container. For wash (1), pour 20 ml of 2X SSC, 0.1% SDS into the plastic container and place in orbital shaker for 5 minutes at room temperature. After the times up, remove the buffer from the plastic container. Repeat wash (1) for another 5 minutes. For wash (2), add 20 ml of 1X SSC, 0.1% SDS into the plastic container and place in orbital shaker for 5 minutes at room temperature. After the times up, remove the buffer from the plastic container. Repeat wash (2) for another 5 minutes. For wash (3), pour 20 ml of 0.1M Maleic acid, 0.15M NaCl pH 7.5, 0.3% Tween 20 into the plastic container and place in orbital shaker for 5 minutes at room temperature. After the times up, remove the buffer from the plastic container. For blocking step, add 20 ml of 1:10 blocking solution into the plastic container and place in orbital shaker for 30 minutes at room temperature. After the times up, remove the buffer from the plastic container.
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Add 10 ml of Anti-digoxigenin conjugated alkaline phosphatase antibody into the plastic container and place in orbital shaker for 30 minutes at room temperature. After the time is up, load the antibody back into the 15 ml Falcon tube. Wash with the same step as in wash (3) with the same reagent, lace in orbital shaker for 5 minutes at room temperature. After the times up, remove the buffer completely. Repeat wash (3) for another 5 minutes. For detection, add 20 ml of detection buffer 0.1M Tris-HCl, 0.1M NaCl, pH 9.5 for 5 minutes at room temperature. The plastic container is cover with an aluminium foil for incubation in dark area while carefully to not shake the plastic container. After the incubation is done, remove the buffer. Add 10 ml of colour substrate solution and incubate for 10 to 40 minutes. Incubate at dark condition and carefully not to shakes the plastic container. After the colour of precipitate is formed or after 40 minutes, remove the solution in the plastic container. Add 20 ml of TE buffer and briefly shake before removing the buffer. Repeat with washing by using TE buffer. Photograph the image of the nitrocellulose membrane.
841009-14-5879 2. The flow chart of method
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RESULT 1. Image of nitrocellulose membrane
Figure 3 shows no band is forming on the nitrocellulose membrane. 3. Result description: There is no band forming on the nitrocellulose membrane after the enzymatic reaction of alkaline phosphatase enzyme + NBT/BCIP. From the 5 groups which attend the same laboratory practical, none of the group has colour band on their nitrocellulose membrane. The main suspected culprit is maybe cause by the failure of the DNA transfer from agarose gel to the nitrocellulose membrane through the Southern blotting technique that take 12 hours to run from the practical 2, which end at 12 midnight and assume at 12 noon for practical 3.
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DISCUSSION 1. The experiment that was carried out uses the principle of enzymatic reaction (alkaline phosphatase enzyme + NBT/BCIP) which produces bluish colour. Draw TWO (2) examples of detection methods other than enzymatic reaction and briefly explain of each method. Example 1 : Chemiluminescent detection of bound DNA probe
The probe was labelled with biotin and binds to target DNA on nitrocellulose membrane. Streptavidin bound to biotin probe, while Alkaline phosphatase conjugated to biotin also binds to Streptavidin. When substrate is added the product will emitting lights and it can be capture using x-ray.
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Example 2: Fluorescent labelled DNA probe
The principle is the same with the enzymatic reaction but instead of NBT/BCIP substrate it used a fluorescein label that can be viewed using fluorescence microscope. The led will excite the fluorescein label causing it to emitted green fluorescence
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2. The hybridized probes are immunodetected with anti-digoxigenin-AP, Fab fragments. Draw the Fab fragment and full-length IgG antibody structure and briefly compared both
Fab fragment is derived from the FAB region of the IgG antibody. It consists of a heavy and light chain and a variable and constant region. The variable region will bind to an epitope of antigen 3. NBT and BCIP stand for what? State the principle of colour development and explain the roles play by NBT and BCIP in the process. NBT and BCIP is a soluble substrate where NBT stand for nitrobluetetrazolium and BCIP stand for 5-bromo-5-chloro-3-indolyl phosphate. The principle is very specific like lock and key. When DNA immobilized on the nitrocellulose membrane is hybridise with a DIG labelled probes, an anti-digoxigenin antibody conjugated alkaline phosphatase will bound to it through the enzymatic reaction forming a complex which then bind the NBT/BCIP substrate. The reaction produces a bluish purple colour precipitate that is visible to the naked eye.
