当前位置:皇家赌场号hj85 > 生命科学 > PCLacrosse方法检查实验Alu重复连串

PCLacrosse方法检查实验Alu重复连串

文章作者:生命科学 上传时间:2019-08-27

In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes that exhibit a great deal of diversity. Such variable sequences are termed "polymorphic" and provide the basis for genetic disease diagnosis, forensic identification, and paternity testing.

Reagents10% Chelex 0.9% sodium chloride PCR reaction mix 25 mM MgCl2 mineral oil DNA size markers distilled water loading dye 1.5% agarose 1X Tris/Borate/EDTA buffer 1 micrograms/ml ethidium bromide For Decontamination:0.05 M KMnO4 0.25 N HCl 0.25 N NaOH Equipment and Supplies100-1000 microliter micropipettor tips 10-100 microliter micropipettor tips 0.5-10 microliter micropipettor tips 15 ml culture tube 1.5 ml tubes 0.5 ml PCR tubes beaker for waste/used tips boiling water bath DNA thermal cycler forceps paper cup Procedure Use permanent marker to label your name on a 15-ml culture tube containing saline solution.

For Decontamination:

  • 0.05 M KMnO4
  • 0.25 N HCl
  • 0.25 N NaOH

In order to compare the genotypes from a number of different individuals, aliquots of the amplified sample and those of other experimenters are loaded into wells of an agarose gel--along with the DNA size markers and an unamplified control. Following electrophoresis and staining, amplification products appear as distinct bands in the gel--the distance moved from the well is inversely proportional to the presence or absence of the TPA-25 insertion. One or two bands are visible in each lane, indicating that an individual is either homozygous or heterozygous for the Alu insertion.

Securely close cap, and place mouthwash tube in a balanced configuration with other tubes in rotor of clinical centrifuge. Centrifuge at 500-1,000 x g for 10 minutes to pellet cells on the bottom side of culture tube.

The Alu family of short interspersed repeated DNA elements are distributed throughout primate genomes. Over the past 65 million years, the Alu sequence has amplified via an RNA-mediated transposition process to a copy number of about 500,000--comprising an estimated 5% of the human genome. Alu sequences are thought to be derived from the 7SL RNA gene which encodes the RNA component of the signal recognition particle that functions in protein synthesis. Alu elements are approximately 300-bp in length and derive their name from a single recognition site for the endonuclease Alu 1 located near the middle of the Alu sequence.

核心提示:In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look

Transfer 200 microliters of the supernatant to a fresh 1.5 ml tube labeled with your name, and place tube on ice. Avoid transferring any of the Chelex pellet.

Procedure

  1. Use permanent marker to label your name on a 15-ml culture tube containing saline solution.
  2. Pour all of the saline solution into your mouth, and vigorously swish for 10 seconds. Save the empty 15-ml tube for reuse in the next step.
  3. Expel saline mouthwash into a paper cup. Then carefully pour saline mouthwash from paper cup back into 15-ml tube from Step 1.
  4. Securely close cap, and place mouthwash tube in a balanced configuration with other tubes in rotor of clinical centrifuge. Centrifuge at 500-1,000 x g for 10 minutes to pellet cells on the bottom side of culture tube.
  5. Being careful not to disturb cell pellet, pour off as much supernatant as possible into sink or paper cup. Place tube with mouthwash cell pellet on ice.
  6. Use micropipettor to add 500 microliters of 10% Chelex to cell pellet:
  7. Resuspend cells in the Chelex by pipetting up and down several times. Hold tube up to light to confirm that no visible clumps of cells remain.
  8. Transfer 500 microliters of the resuspended mouthwash sample into a clean 1.5 ml reaction tube labeled with your name.
  9. Incubate the 1.5 ml sample tube in a boiling water bath for 10 minutes.
  10. Following incubation, use forceps to remove sample tube from boiling water bath, and cool tube on ice for approximately one minute.
  11. Place sample tube in a balanced configuration in a microfuge rotor, and spin for 30 seconds to pellet Chelex beads at bottom of tube.
  12. Transfer 200 microliters of the supernatant to a fresh 1.5 ml tube labeled with your name, and place tube on ice. Avoid transferring any of the Chelex pellet.
  13. Set up PCR reaction using 5 microliters of DNA sample in a 50 microliter reaction volume.
  14. Amplify using the following program:
  15. Electrophorese 10 microliters of the PCR reaction in a 1.5% agarose gel, stain, view, and photograph.

In this experiment, oligonucleotide primers, flanking the insertion site, are used to amplify a 400-bp fragment when TPA-25 is present and a 100-bp fragment when it is absent. Each of the three possible genotypes--homozygotes for presence of TPA-25 , homozygous for absence of TPA-25 , and heterozygotes are distinguished following electrophoresis in agarose gels.

The Alu family of short interspersed repeated DNA elements are distributed throughout primate genomes. Over the past 65 million years, the Alu sequence has amplified via an RNA-mediated transposition process to a copy number of about 500,000--comprising an estimated 5% of the human genome. Alu sequences are thought to be derived from the 7SL RNA gene which encodes the RNA component of the signal recognition particle that functions in protein synthesis. Alu elements are approximately 300-bp in length and derive their name from a single recognition site for the endonuclease Alu 1 located near the middle of the Alu sequence.

In order to compare the genotypes from a number of different individuals, aliquots of the amplified sample and those of other experimenters are loaded into wells of an agarose gel--along with the DNA size markers and an unamplified control. Following electrophoresis and staining, amplification products appear as distinct bands in the gel--the distance moved from the well is inversely proportional to the presence or absence of the TPA-25 insertion. One or two bands are visible in each lane, indicating that an individual is either homozygous or heterozygous for the Alu insertion.

For Decontamination:

  • 0.05 M KMnO4
  • 0.25 N HCl
  • 0.25 N NaOH

皇家赌场号hj85,Pour all of the saline solution into your mouth, and vigorously swish for 10 seconds. Save the empty 15-ml tube for reuse in the next step.

An estimated 500-2,000 Alu elements are mostly restricted to the human genome. A few of these have inserted recently, within the last one million years, and are not fixed in the human species. One such Alu element, called TPA-25, is found within an intron of the tissue plasminogen activator gene. This insertion is dimorphic, meaning that it is present in some individuals and not in others. PCR can be used to screen individuals for the presence of the TPA-25 insertion.

Procedure

  1. Use permanent marker to label your name on a 15-ml culture tube containing saline solution.
  2. Pour all of the saline solution into your mouth, and vigorously swish for 10 seconds. Save the empty 15-ml tube for reuse in the next step.
  3. Expel saline mouthwash into a paper cup. Then carefully pour saline mouthwash from paper cup back into 15-ml tube from Step 1.
  4. Securely close cap, and place mouthwash tube in a balanced configuration with other tubes in rotor of clinical centrifuge. Centrifuge at 500-1,000 x g for 10 minutes to pellet cells on the bottom side of culture tube.
  5. Being careful not to disturb cell pellet, pour off as much supernatant as possible into sink or paper cup. Place tube with mouthwash cell pellet on ice.
  6. Use micropipettor to add 500 microliters of 10% Chelex to cell pellet:
  7. Resuspend cells in the Chelex by pipetting up and down several times. Hold tube up to light to confirm that no visible clumps of cells remain.
  8. Transfer 500 microliters of the resuspended mouthwash sample into a clean 1.5 ml reaction tube labeled with your name.
  9. Incubate the 1.5 ml sample tube in a boiling water bath for 10 minutes.
  10. Following incubation, use forceps to remove sample tube from boiling water bath, and cool tube on ice for approximately one minute.
  11. Place sample tube in a balanced configuration in a microfuge rotor, and spin for 30 seconds to pellet Chelex beads at bottom of tube.
  12. Transfer 200 microliters of the supernatant to a fresh 1.5 ml tube labeled with your name, and place tube on ice. Avoid transferring any of the Chelex pellet.
  13. Set up PCR reaction using 5 microliters of DNA sample in a 50 microliter reaction volume.
  14. Amplify using the following program:
  15. Electrophorese 10 microliters of the PCR reaction in a 1.5% agarose gel, stain, view, and photograph.

Amplify using the following program:

Reagents

  • 10% Chelex
  • 0.9% sodium chloride
  • PCR reaction mix
  • 25 mM MgCl2
  • mineral oil
  • DNA size markers
  • distilled water
  • loading dye
  • 1.5% agarose
  • 1X Tris/Borate/EDTA buffer
  • 1 micrograms/ml ethidium bromide

Equipment and Supplies

  • 100-1000 microliter micropipettor tips
  • 10-100 microliter micropipettor tips
  • 0.5-10 microliter micropipettor tips
  • 15 ml culture tube
  • 1.5 ml tubes
  • 0.5 ml PCR tubes
  • beaker for waste/used tips
  • boiling water bath
  • DNA thermal cycler
  • forceps
  • paper cup

a. Pipet Chelex solution in and out of pipet tip several times to suspend the Chelex beads.

核心提示:In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look

The source of template DNA is a sample of several thousand cells obtained by saline mouthwash . The cells are collected by centrifugation and resuspended in a solution containing the resin "Chelex," which binds metal ions that inhibit the PCR reaction. The cells are lyzed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing genomic DNA is mixed with Taq polymerase, olignucleotide primers, the four deoxyynucleotides, and the cofactor magnesium chloride. Temperature cycling is used to denature the target DNA, anneal the primers, and extend a complementary DNA strand. The "upstream" primer, "5-GTAAGAGTTCCGTAACAGGACAGCT-3", brackets one side of the TPA locus, while the "downstream" primer, "5-CCCCACCCTAGGAGAACTTCTCTTT-3" brackets the other side. The size of the amplification product depends on the presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8.

核心提示:In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes that exhibit a great deal of diversity. Such variable sequences are termed "polymorphic" and provide the basis for genetic disease diagnosis, forensic identification, and paternity testing.

Equipment and Supplies

  • 100-1000 microliter micropipettor tips
  • 10-100 microliter micropipettor tips
  • 0.5-10 microliter micropipettor tips
  • 15 ml culture tube
  • 1.5 ml tubes
  • 0.5 ml PCR tubes
  • beaker for waste/used tips
  • boiling water bath
  • DNA thermal cycler
  • forceps
  • paper cup

The Alu family of short interspersed repeated DNA elements are distributed throughout primate genomes. Over the past 65 million years, the Alu sequence has amplified via an RNA-mediated transposition process to a copy number of about 500,000--comprising an estimated 5% of the human genome. Alu sequences are thought to be derived from the 7SL RNA gene which encodes the RNA component of the signal recognition particle that functions in protein synthesis. Alu elements are approximately 300-bp in length and derive their name from a single recognition site for the endonuclease Alu 1 located near the middle of the Alu sequence.

b. Before the Chelex has a chance to settle, transfer the 500 microliters to the culture tube.

In this experiment, polymerase chain reaction is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes that exhibit a great deal of diversity. Such variable sequences are termed "polymorphic" and provide the basis for genetic disease diagnosis, forensic identification, and paternity testing.

Reagents

  • 10% Chelex
  • 0.9% sodium chloride
  • PCR reaction mix
  • 25 mM MgCl2
  • mineral oil
  • DNA size markers
  • distilled water
  • loading dye
  • 1.5% agarose
  • 1X Tris/Borate/EDTA buffer
  • 1 micrograms/ml ethidium bromide

Expel saline mouthwash into a paper cup. Then carefully pour saline mouthwash from paper cup back into 15-ml tube from Step 1.

The source of template DNA is a sample of several thousand cells obtained by saline mouthwash . The cells are collected by centrifugation and resuspended in a solution containing the resin "Chelex," which binds metal ions that inhibit the PCR reaction. The cells are lyzed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing genomic DNA is mixed with Taq polymerase, olignucleotide primers, the four deoxyynucleotides, and the cofactor magnesium chloride. Temperature cycling is used to denature the target DNA, anneal the primers, and extend a complementary DNA strand. The "upstream" primer, "5-GTAAGAGTTCCGTAACAGGACAGCT-3", brackets one side of the TPA locus, while the "downstream" primer, "5-CCCCACCCTAGGAGAACTTCTCTTT-3" brackets the other side. The size of the amplification product depends on the presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8.

An estimated 500-2,000 Alu elements are mostly restricted to the human genome. A few of these have inserted recently, within the last one million years, and are not fixed in the human species. One such Alu element, called TPA-25, is found within an intron of the tissue plasminogen activator gene. This insertion is dimorphic, meaning that it is present in some individuals and not in others. PCR can be used to screen individuals for the presence of the TPA-25 insertion.

In order to compare the genotypes from a number of different individuals, aliquots of the amplified sample and those of other experimenters are loaded into wells of an agarose gel--along with the DNA size markers and an unamplified control. Following electrophoresis and staining, amplification products appear as distinct bands in the gel--the distance moved from the well is inversely proportional to the presence or absence of the TPA-25 insertion. One or two bands are visible in each lane, indicating that an individual is either homozygous or heterozygous for the Alu insertion.

In this experiment, oligonucleotide primers, flanking the insertion site, are used to amplify a 400-bp fragment when TPA-25 is present and a 100-bp fragment when it is absent. Each of the three possible genotypes--homozygotes for presence of TPA-25 , homozygous for absence of TPA-25 , and heterozygotes are distinguished following electrophoresis in agarose gels.

In this experiment, oligonucleotide primers, flanking the insertion site, are used to amplify a 400-bp fragment when TPA-25 is present and a 100-bp fragment when it is absent. Each of the three possible genotypes--homozygotes for presence of TPA-25 , homozygous for absence of TPA-25 , and heterozygotes are distinguished following electrophoresis in agarose gels.

Electrophorese 10 microliters of the PCR reaction in a 1.5% agarose gel, stain, view, and photograph.

Incubate the 1.5 ml sample tube in a boiling water bath for 10 minutes.

58 degrees C for 2 minutes

Transfer 500 microliters of the resuspended mouthwash sample into a clean 1.5 ml reaction tube labeled with your name.

Place sample tube in a balanced configuration in a microfuge rotor, and spin for 30 seconds to pellet Chelex beads at bottom of tube.

Resuspend cells in the Chelex by pipetting up and down several times. Hold tube up to light to confirm that no visible clumps of cells remain.

Following incubation, use forceps to remove sample tube from boiling water bath, and cool tube on ice for approximately one minute.

An estimated 500-2,000 Alu elements are mostly restricted to the human genome. A few of these have inserted recently, within the last one million years, and are not fixed in the human species. One such Alu element, called TPA-25, is found within an intron of the tissue plasminogen activator gene. This insertion is dimorphic, meaning that it is present in some individuals and not in others. PCR can be used to screen individuals for the presence of the TPA-25 insertion.

Set up PCR reaction using 5 microliters of DNA sample in a 50 microliter reaction volume.

94 degrees C for 1 minute

Being careful not to disturb cell pellet, pour off as much supernatant as possible into sink or paper cup. Place tube with mouthwash cell pellet on ice.

72 degrees C for 2 minutes 30 cycles

Use micropipettor to add 500 microliters of 10% Chelex to cell pellet:

The source of template DNA is a sample of several thousand cells obtained by saline mouthwash . The cells are collected by centrifugation and resuspended in a solution containing the resin "Chelex," which binds metal ions that inhibit the PCR reaction. The cells are lyzed by boiling and centrifuged to remove cell debris. A sample of the supernatant containing genomic DNA is mixed with Taq polymerase, olignucleotide primers, the four deoxyynucleotides, and the cofactor magnesium chloride. Temperature cycling is used to denature the target DNA, anneal the primers, and extend a complementary DNA strand. The "upstream" primer, "5-GTAAGAGTTCCGTAACAGGACAGCT-3", brackets one side of the TPA locus, while the "downstream" primer, "5-CCCCACCCTAGGAGAACTTCTCTTT-3" brackets the other side. The size of the amplification product depends on the presence or absence of the Alu insertion at the TPA-25 locus on each copy of chromosome 8.

本文由皇家赌场号hj85发布于生命科学,转载请注明出处:PCLacrosse方法检查实验Alu重复连串

关键词: 皇家赌场号hj85