CLONING OF FACTOR VIII

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CLONING SIMULATION PROJECT

• CLONING OF FACTOR VIII
• GROUP C

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THE MEMBERS

• JAIME JACQUELINE
• KHAIRULMAZIDAH MOHAMED
• LELYAN MINSUN
• CHOT SAN NGAN
• CHE SHURAYA CHE ISMAIL
• HASLINDA BT MOHD FAUZI
• MOHD ADIB BIN YAHYA
• MOHD AFIF BIN MOHD NASIR

3
OBJECTIVES

• To understand the concept of molecular biology
  and the application of recombinant DNA technology
  introduced in GTB204/3- Molecular Biology
  Technique.
• To obtain the experience in cloning strategy
  through simulation.
• To expose ourselves to the finding from the
  concerned website such as Bioedit, Genbank,
  CLUSTALW and BLAST and analyze the information as
  well as organize our work to get the final
  product.
• To practice cooperation and knowledge sharing
  among group members.

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Project background

• We agree to choose factor VIII, a type of blood
  clotting factor to be cloned for our cloning
  simulation project
• We compare the sequence using BLAST analysis and
  multiple DNA sequence alignment through the
  CLUSTALW program
• We organize the whole steps we need to get our
  gene of interest
• We design primer and probe for PCR amplification
  clone screening and DNA sequencing as well as
  choosing the appropriate expression vector
• The cloning strategy is designed
• PCR are done to amplify our gene sequence before
  it is inserted into the vector and transformed
  into the host cells.
• The recombinant protein was then expressed (IPTG
  induction).
• Extraction and purification of targeted protein
  was further carried out using Immobilized Metal
  Affinity Chromatography (IMAC).

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INTRODUCTION

• Factor VIII is a protein that is one of the 13
  factors in the blood involved in the bodys
  ability to form clots and to stop bleeding
• A deficiency in clotting Factor VIII causes
  hemophilia A that results in abnormal bleeding
• Small wounds and puncture are normally not a
  problem but uncontrolled internal bleeding can
  result in pain, swelling and permanent damage,
  especially to joints and muscles

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The effect of factor VIII on the blood clotting
mechanism
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What Is Hemophilia A?

• Hemophilia A is caused by an inherited sex-linked
  recessive trait with the defective gene located
  on the X chromosome
• Females are carriers of this trait. Fifty percent
  of the male offspring of female carriers have the
  disease and 50 of their female offspring are
  carriers
• All female children of a male with hemophilia are
  carriers of the trait.

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Hereditary diagram of hemophilia A
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Symptoms

• Bruising
• Spontaneous bleeding
• Bleeding into joints and associated pain and
• swelling
• Gastrointestinal tract and urinary tract
• hemorrhage
• Blood in the urine or stool
• Prolonged bleeding from cuts, tooth extraction,
• and surgery

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Restriction enzymes analysis -restriction enzymes
that cut the sequence NdeI and XhoI
Polymerase chain reaction (PCR)
amplification -Designing primers -Determine
annealing temperature 66oC -Enzyme Taq DNA
polymerase
Adjoining linkers -designing linkers
Choosing suitable vector -expression vector pET
16b

• Restriction of vector and gene sequence of
  interest
• using restriction enzymes NdeI and XhoI
• Produce sticky ends

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Vector treatment -calf intestinal alkaline
phosphate (CIAP) -Prevent self-ligation
Ligation Enzyme T4 DNA ligase
Transformation -host BL21 (DE3) -Method
calcium chloride method
Screening-selection of bacterial host cells
BL21(DE3) -selection for antibiotic resistant
colonies on LB ampicilin agar plate
Patching the colonies
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Plasmid purification -mini plasmid preparation
method
Expression of protein of interest -IPTG acts as
inducers to initiate the transcription and
translation of factor VIII gene
Bacterial lysis -modified version of high salt
buffer 8M urea method
Protein purification -IMAC
-SDS-PAGE
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STEP 1 GENE SEARCH

• We search for the data of the gene we wanted,
  factor VIII through the gene bank web site at
  http//www.ncbi.nlm.nih.gov.
• The accession number for the gene is BC022513
• The gene sequence have 2536bp but the gene that
  produce factor VIII protein are coded from
  64-715bp
• Gene-homo sapiens coagulant factor VIII,
  procoagulant (hemophilia A)

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GENE SEQUENCE
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STEP 2 OPEN READING
FRAME(ORF)

• Our gene start with codon ATG(Met) and end with a
  stop codon TAG
• This analysis is to ensure that our interested
  gene region, that is from 65-715bp can be
  expressed if it is to be cloned in real life
  application
• Go to this website
• http//www.ncbinlm.nih.gov/gorf/gorf.html by
  entering the accession number of BC022513
• 
  

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ORF ANALYSIS RESULT
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OPEN READING FRAME
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STEP 3 DETERMINING MOLECULAR WEIGHT OF FACTOR
VIII GENE

• The molecular weight of the gene is
• (648/3) x 110 23760 Daltons
• approximately 24 kD

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STEP4COMPARE THE GENE SEQUENCE

• BLAST ANALYSIS
• Comparison of the gene sequence with other
  similar gene sequence is done by using BLAST
• Analysis and multiple DNA sequence alignment
• BLAST(Basic Local Alignment Search Tool) is a
  alogarithm used by a family of five programs that
  will align query sequence against sequences in a
  molecular database

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Distribution of 65 Blast Hits on the Query
Sequence
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MULTIPLE DNA SEQUENCE ALIGNMENT

• Capable of comparing the gene with several other
  genes simultaneously.
• Done using the CLUSTALW program.
• Several gene sequence to be compare with factor
  VIII is selected from the Blast result
• We have choosen 4 different sequences
  (U49517,NM-007977,AF 049489 and NM-000132) to
  compare for the similarity

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THE RESULT
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The tree view among the genes being compared with
factor VIII gene
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STEP 5 RESTRICTION ENZYME ANALYSIS

• To analyze the restriction enzyme that will cut
  the gene sequence
• Go to the website
• http//www.firstmarker.com/cutter/cut2.html
• Restriction enzymes that cut in the middle of the
  strand must be excluded to avoid the production
  of unwanted peptides

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RESULT

• The following restriction enzymes does not cut
  the gene sequence of factor VIII

AatI, AatII, Acc113I, Acc16I, Acc65I, AccB1I,
AccB7I, AccBSI, AccI, AccIII, AclNI, AcyI, AfeI,
AflII, AflIII, AgeI, AhdI, AlwNI, Ama87I,
Aor51HI, ApaI, AscI, AseI, AsnI, Asp718I, AspEI,
AspI, AtsI, AvaI, AviII, AvrII, BanI, BanII,
BanIII, BbeI, BbiII, BbrPI, BcgI, BclI, BcoI,
BfrI, BglI, BglII, BlnI, BlpI, Bpu1102I, Bsa29I,
BsaAI, BsaHI, BsaOI, BsaWI, BscI, Bse118I, BseAI,
BseCI, BsePI, BsgI, Bsh1285I, BshNI, BsiEI, BsiI,
BsiMI, BsiWI, BsmBI, BsoBI, Bsp106I, Bsp120I,
Bsp13I, Bsp1407I, Bsp143II, Bsp1720I, Bsp19I,
Bsp68I, BspCI, BspDI, BspEI, BspHI, BspLU11I,
BspMI, BspTI, BspXI, BsrBI, BsrFI, BsrGI, BssAI,
BssHII, BssSI, Bst1107I, Bst98I, BstD102I,
BstDSI, BstEII, BstH2I, BstMCI, BstPI, BstSFI,
BstSNI, BstZI, Bsu15I, CciNI, CelII, Cfr10I,
Cfr42I, Cfr9I, ClaI, CpoI, CspI, DraIII, DrdI,
DsaI, EagI, Eam1105I, Ecl136II, EclHKI, EclXI,
Eco105I, Eco147I, Eco24I, Eco255I, Eco32I,
Eco47III, Eco52I, Eco57I, Eco64I, Eco72I,
Eco88I,Eco91I, EcoICRI, EcoNI, EcoO65I, EcoRV,
EcoT22I, EheI, Esp1396I, Esp3I, FauNDI, FbaI,
FriOI, FseI, FspI, HaeII, Hin1I, HincII, HindII,
HindIII, HpaI, Hsp92I, KasI, Kpn2I, KpnI, Ksp22I,
KspI, MfeI, MluI, Mph1103I, MroI, MroNI, Msp17I,
MspA1I, MspCI, MunI, NaeI, NarI, NcoI, NdeI,
NgoAIV, NgoMI, NheI, NotI, NruI, NsiI,
NspBII,PacI, PaeR7I, Pfl23II, PflMI, PinAI,
Ple19I, PmaCI, Pme55I, PmeI, PmlI, Ppu10I,
PshAI, PshBI, Psp124BI, Psp1406I, PspAI, PspALI,
PspEI, PspLI, PspOMI, PstI, PstNHI, PvuI, PvuII,
RcaI, RsrII, SacI, SacII, SalI, SapI, SbfI, ScaI,
SfcI, SfiI, Sfr274I, Sfr303I, SgfI, SgrAI, SmaI,
SnaBI, SpeI, SplI, SrfI, Sse8387I, SseBI, SspBI,
SstI, SstII, StuI,SunI, Tth111I, Van91I, Vha464I,
VspI, XcmI, XhoI, XmaI, XmaIII, Zsp2I
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• we will choose Nde I and Xho I as our restriction
  enzymes

• produce sticky ends site which can base pair with
  complementary ends

• The restriction site recognized by Nde I and Xho
  I are as shown below

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STEP 6 PCR AMPLIFICATION

• It is an in vitro technique for the amplification
  of a region of DNA which lies between two regions
  of known DNA sequence.
• We use oligonucleotide primers
• a rapid and efficient way to replicate specific
  fragments of DNA.

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DESIGNING PRIMER

• Primers are typically short, single stranded
  oligonucleotide (DNA) which are complementary to
  the other regions of known sequence.
• usually between 18-30 mer (bases) in length and
  has 35 to 65 GC content
• Invert repeated sequence should be avoided as it
  will prevent hybridization to the template.
• Forward primer should not be complementary to
  reverse primer
• 3 end of the primer should be complementary to
  the target DNA sequence, 5 end of the primer can
  have other sequences like restriction enzyme
  sites, promoter sites, etc. the distance between
  primers is preferably less than 10kb in length.
• we design our own forward and reverse primers.
  The primers we are using have 21 bp.
• Forward primer 5
  ATGCGGATCCAAGACCCTGGG 3
• Reverse primer 5
  CCCTCAGTAGAGGTCCTGTGC 3

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Forward primer 5 ATGCGGATCCAAGACCCTGGG 3
A 5 T 3 C
6 G 7 Tm (A
T) 2 (G C)4
(5 3)2 (7 6)4
68ºC.
Annealing temperature 68ºC - 2ºC

66ºC. Reverse primer 5 CCCTCAGTAGAGGTCCTGT
GC 3 A 3
T 5 C 7 G 6
Tm (A T) 2 (G C) 4
(3 5)2 (7 6) 4
68ºC.
Annealing
temperature 68ºC - 2ºC

66ºC.
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• Both forward and reverse primers have the same
  annealing temperature

• We run our PCR at 66ºC

• Other components needed for PCR amplification

• Taq DNA polymerase
• primers
• template
• KCl 50mM
• Tris 10mM at pH 8.3
• MgCl 1.5mM
• dNTPs 0.2mM each dNTP (dATP,dCTP,dGTP,dTTP)

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• One cycle consisting of

• an initial denaturing step of 95? C for 4 minutes,

• 30 cycle consisting of denaturation at 95?C for
  30 seconds, annealing at 66?C for 30 seconds and
  extention at 72?C for 30 seconds .

• Annealing at 66?C for 1 minute and extention at
  72?C for 5 minutes

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STEPS IN PCR

• Template denaturation
• accomplished at 95?C to 100?C
• Primer annealing temperature
• Each primer has its own characteristic annealing
  temperature
• Its length and base composition as well as the
  reaction buffer ionic strength are taken account
• The empirically determined annealing temperature
  are more ideal than the calculateds.
• Primer extension
• performed at 72?C
• we run the PCR product on the agarose gel for
  electrophoresis.
• PCR product was visualized under the UV light.
• From the electrophoresis result, we can detect
  the gene sequence of interest that have been
  amplified

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OVERVIEW

• Choosing a gene of interest from Genebank
• BC022513
• Homo sapiens coagulation factor VIII, proagulant
• 2536 base pairs

Open reading frame analysis by using Genebank
website The 1 frame that includes the whole
frame from base pair 65 to 175, which has the
length of 651bp are selected
Determining amino acid sequence and molecular
weight - the MW approximately 24kD
Blast analysis -reference code
1061259619-22741-1117648
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STEP7 ADJOINING A LINKER TO FACTOR VIII
INSERT

• Linkers are small self-complementary pieces of
  synthetic DNA
• Usually 8-16 nucleotides in length, that anneal
  to form blunt ends, double-stranded molecules
  containing a recognition site for a restriction
  enzyme

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• We design our own linkers

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STEP 8 CHOOSING SUITABLE VECTOR

• We have chosen pET-16b (expression vector)
• The vector
• highest expression levels and highest control
  over basal expression
• has precise control of induced expression with
  IPTG
• has the choice of N-terminal and C-terminal
  fusion tags for detection, purification and
  localization as well as expanded multiple cloning
  sites plus f1 origin of replication for
  mutagenesis and sequencing.
• has convenient restriction sites for subcloning
  from other vectors and to purify the target
  proteins
• has ampicilin resistance marker, a gene that
  resistant to antibiotic ampicilin
• Carries an N-terminal his-tag sequence followed
  by a factor Xa site and 3 cloning sites

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STEP 9 RESTRICTION OF VECTOR AND
GENE

• The pET16b vector and the gene fragment of
  interest (insert) are digested with the same
  restriction enzymes, NdeI and XhoI.
• We are doing the directional cloning
• DNA fragments is produced by digestion with two
  restriction enzymes with different recognition
  sequences
• Both the enzymes will cut at the palindromic
  sequence of the vector and produce sticky end at
  both sides.

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STEP11 LIGATION

• A process to insert and join our DNA fragment
  into our vector (pET 16b )
• We used
• -T4 DNA ligase
• catalyses the formation of
  phosphodiester bond
• between 3 hydroxyl and 5phosphate
• -Ligase buffer
• -Sterile deionised water
• Sticky ends are more efficiently joined compared
  to blunt ends.

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Step 12 TRANSFORMATION

• Transfer of genetic information by means of
  extracellular piece of DNA to the bacterial host.
• Competent cell- able to take up foreign DNA from
  external environment
• Host -E. Coli BL21(DE3).
• Genetically engineered vector designated
  exclusively for eukaryote protein expression in
  prokaryote system.
• Not naturally competent- calcium chloride method.

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• TRANSFORMATION - using calcium chloride method

Our Host culture (E.coli) are pelleted by
centrifugation ? bacterial pellet is resuspended
into CaCl2 solution ? Incubated in an ice water
bath ? competent cell store at 80C
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Ligation mix is added into the competent
cell ? We keep it on ice for 2 minutes
?(heat shock) We keep the tube on 42C
water bath 30 second ? Keep on ice again 2
minutes ? Immediately we add transformation
product to the plate (LB ampicillin)
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• During transformation reaction

• Competent cells are combined with the ligation
  product.
• Calcium Chloride causes the DNA to
  precipitate onto the outside of the cell
• Incubation on ice plasmid DNA sticks to the
  outer cell walls of the membrane
• Heat shock to make membranes of the E.coli
  become more porous and allow DNA to enter into
  the host

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STEP13 SCREENING-SELECTION FOR
ANTIBIOTIC RESISTANT COLONIES

• Transformation efficiency of plasmids in E.coli
  is very low
• Most E.coli cells that proliferate in the medium
  would not contain the plasmids
• Selection of bacteria cells or E.Coli need to be
  done
• We plate our culture on LB Ampicillin agar plates
• This selective antibiotic is that E. coli is
  sensitive to ampicillin in nature and will not
  grow on ampicillin plate without the presence of
  antibiotic resistance marker
• Our bacterial colonies on the plate had taken up
  the pET-16b plasmid which made them antibiotic
  resistant.

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• The transformed E. coli cells are protected by
  the ampicillin- resistance gene on the plasmids,
  which can express the enzyme, ß-lactamase to
  inactivate the antibiotic ampicillin
• By this, we can select our transformed cells for
  screening method.
• After an overnight incubation at 37ºC of the
  culture, we found out there are colonies present
  in the LB ampicillin agar plate
• Although colonies are present in the LB
  ampicillin agar plate, we cannot determine which
  bacterial colony has the recombinant DNA
• We choose an alternative to detect and check our
  insert by plasmid purification in the agarose gel
  electrophoresis. Before the plasmid purification
  step, we need to patch the colonies first.

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Step 14 PATCHING THE COLONIES

• Transformation the ligation mixture
• ?
• Colonies on LB Ampicillin
• ?
• 4 colonies selected
• Patch on LBA agar plate
• ?
• Streak using mine loop
• ?
• Incubated overnight at 37C
• ?
• Plasmid purification

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STEP 15 PLASMID PURIFICATION

• Purification of high quality plasmid DNA is
  necessary for successful genome sequencing
  project.
• To confirm our successful recombinant DNA
  molecule we have actually extracted the plasmid
  containing the recombinant product from the host
  cell
• We use mini plasmid preparation method to extract
  the plasmid successfully

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Steps in plasmid purification
Plasmid extraction Restriction enzyme
digestion Electrophoresis gel analysis
53

• As a result, we see two fragments on both vector
  and the insert in length, ranging approximately
  636bp and 5100bp in the test lane.
• The marker that we used was ?Hind III (bp ladder)

• This shown that the insert was successfully
  inserted into the plasmid in our previous step.
  Now, we may proceed with protein expression by
  choosing the appropriate colonies

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STEP 16 EXPRESSION

• Recombinant culture is prepared overnight
• ?
• It was cultured again and incubated for 3 hours.
• ?
• Induced using 0.4 mM IPTG
• ?
• Grown for another 3 hours
• ?
• The culture is spinned
• ?
• Pellet bacterial cell produced (has protein of
  interest)
• ?
• The bacteria was lysed in next step

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STEP 17 BACTERIAL LYSIS

• to get protein that was expressed by the
  bacteria.
• modified version of high salt buffer 8.0M urea
  method.
• buffer - 2 D lysis buffer

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STEP 18 PROTEIN PURIFICATION
Supernatant recombinant protein ? Pass it
through a nickel affinity column ? Wash the
unbound protein or protein without the His-Tag
with buffer ? Elute the recombinant protein with
His-Tag by using the imidazole ? His-Tag protein
is treated with the specific protease to cleave
off the His-Tag ? The recombinant protein is
freed of the His-Tag peptide by running it over
the metal chelate column again
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STEP 19 SDS-PAGE

• To make sure the pure protein is purify.
• By comparing to the molecular weight marker.
• Approximately 24kD
• Now the pure purified protein is ready for use

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kDa Molecular weight
induced
uninduced
IMAC
97
67
40
20
14
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Commercial Value of recombinant factor VIII

• Large supply.
• Low cost.
• Avoid viral infection
• - No blood transfusion required.
• - Highly purified recombinant factor viii
• - Eliminate risk of transmission of HIV,HEV

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Conclusion

• Understand most molecular biology concept and
  application of recombinant DNA technology.
• Gain experience in cloning a gene through
  simulation.
• Gain experience in using Bioedit, Genbank,
  CLUSTALW, BLAST, Webcutter website.
• Learned how to analyze the information and
  organize our work.

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REFERENCE

• Genetics The Continuity of LifeDaniel J
  Fairbanks,W.Ralph AndersenWadsworth,1990pg
  255-289.
• Life Science Catalog 2003Promega.
• StratageneTools and Technology for Life Sciences
  2001/2002 Catalog
• Invitrogen 2000 Catalog
• Essential Cell BiologyAlberts,Bray,Johnson,Lewis,
  Raff,Roberts,WalterGarland Publishing Inc,1998.
• http//www.ncbi.nlm.nih.gov
• http//www.rna.lundberg.gn.se./cutter2
• http//www.2.ebi.ac.uk/clustalw
• http//www.genome.wi.mit.edu/cgi_bin/primer/
• http//www.novagen.com/sharedImages/technicalliter
  ature/7_TB046.pdf
• http//www.novagen.com/docs/NOIS/69662-000html
• http//www.novagen.com/sharedImages/technicalliter
  ature/7_pet16bs.htm
• http//www.restools.sdsc.edu/biostools.html
• http//www.abtbeads.com/applications/metal.chelati
  ng.beads.html
• http//www.cne.utexas.edu/nams/NAM597_Abs/characte
  rization/c3.html
• http//www.altcorp.com/affinitylabeling/imac.htm
• http//www.bio-nobile.com
• http//www.perbio.com.cn/PIERCE/technique/antibody
  
• http//www.ucalgary.ca/dnalab/stock primers.htm

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THANK YOU TERIMA KASIH SPACIBA MERCI