IGEM:IMPERIAL/2007/Tutorials/Guide for Engineers/Standard Lab Techniques - Revision history

Alexander.wong: /* Gene Cloning */

Alexander.wong — Sun, 22 Jul 2007 11:46:20 GMT

Gene Cloning

←Older revision		Revision as of 11:46, 22 July 2007
Line 43:		Line 43:

	Cells containing pBR322 with a DNA insert at the SalI site are resistant to ampicillin but sensitive to tetracycline, so they can be readily selected. Cells that failed to take up the vector are sensitive to both antibiotics. Cells that take up pBR322 without a DNA insert are resistant to both. The colony of cells that are shown to have taken up the correct recombinant DNA is grown to produce a large amount of the DNA of interest.		Cells containing pBR322 with a DNA insert at the SalI site are resistant to ampicillin but sensitive to tetracycline, so they can be readily selected. Cells that failed to take up the vector are sensitive to both antibiotics. Cells that take up pBR322 without a DNA insert are resistant to both. The colony of cells that are shown to have taken up the correct recombinant DNA is grown to produce a large amount of the DNA of interest.
		+
		+	<br clear="all">

	== Gel Electrophoresis ==		== Gel Electrophoresis ==

Alexander.wong: /* Recombinant DNA */

Alexander.wong — Sun, 22 Jul 2007 11:45:56 GMT

Recombinant DNA

←Older revision		Revision as of 11:45, 22 July 2007
Line 32:		Line 32:

	The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.		The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.
		+
		+	<br clear="all">

	== Gene Cloning ==		== Gene Cloning ==

Alexander.wong: /* PCR (Polymerase Chain Reaction) */

Alexander.wong — Sun, 22 Jul 2007 11:45:23 GMT

PCR (Polymerase Chain Reaction)

←Older revision		Revision as of 11:45, 22 July 2007
Line 88:		Line 88:

	There are several noteworthy features of this method. First, the sequence of the target need not be known. Only knowledge of the flanking sequences is required. Second, the target can be much larger than the primers. (>10 kb) Third, primers do not have to perfectly match flanking sequences. Primers derived from a gene of known sequence can be used to amplify related genes of the same family. Fourth, stringency or the closeness of the match between primer and target can be controlled by temperature and salt (MgCl2). Fifth, PCR is very sensitive. A single DNA molecule can be amplified and detected.		There are several noteworthy features of this method. First, the sequence of the target need not be known. Only knowledge of the flanking sequences is required. Second, the target can be much larger than the primers. (>10 kb) Third, primers do not have to perfectly match flanking sequences. Primers derived from a gene of known sequence can be used to amplify related genes of the same family. Fourth, stringency or the closeness of the match between primer and target can be controlled by temperature and salt (MgCl2). Fifth, PCR is very sensitive. A single DNA molecule can be amplified and detected.
		+
		+	<br clear="all">

	== DNA Sequencing ==		== DNA Sequencing ==

James Chappell: /* Blotting Techniques */

James Chappell — Sun, 22 Jul 2007 10:23:26 GMT

Blotting Techniques

←Older revision		Revision as of 10:23, 22 July 2007
Line 58:		Line 58:

	== Blotting Techniques ==		== Blotting Techniques ==
-		+	Bloting is the methd of transferring biological molecules onto a carrier such as nitrocellulose membranes. Normally this transfer is after gel electrophoresis to allow for further experiments to be carried out on a separated sample. There are several varieties of blots, which vary in the types of molecules that they separate. The three types are, Southern blotting for DNA, Northern blotting for RNA, and Western blotting for proteins.
-	~~===~~ Western ~~Blotting ===~~	+

	=== Southern Blotting ===		=== Southern Blotting ===
		+	Southern blotting is teh transfer of DNA molecules that have been separated using gel electrophoresis onto a membrane such as nitrocellulose. Once transferred, the DNA molecules can be visualised by probe hybridisation. Hybridisation is the ability of complementary single strands of DNA to form a double helix and this. Hybridisation allows us to probe for specific sequences of DNA, by adding a complementary probe, either RNA or DNA, that have been labelled to allow for visualisation. Labelling types include radioactiveand fluorescent.

-	=== Northern Blotting ===	+	===Northern Blotting ===
		+	Northern blotting is similar to southern blotting, however teh key differece is that it does not involve the transfer of DNA molecules but rather RNA molecules. This method is normally used for testing expression variations, where mRNA samples of a cell are extracted, separated. transferred and visualised. Again for visualisation, labelled DNA or RNA probes are used and hybridised to the sample of RNA.
		+
		+	=== Western Blotting ===
		+	Western blotting is a method that is used to identify protein from a sample. A mixture of protein is separated on either native or denaturing gels and then transferred to a membrane. Specific proteins can then be identified using antibodies that bind to specific proteins. These antibodies are labelled, usually by either fluorescence or radioactivity. In addition, antibodies can be coupled to enzymes such as horseradish peroxidase that allows for detection.

	== PCR (Polymerase Chain Reaction) ==		== PCR (Polymerase Chain Reaction) ==

Cheuk Ka Tong: /* PCR (Polymerase Chain Reaction) */

Cheuk Ka Tong — Mon, 16 Jul 2007 20:32:23 GMT

PCR (Polymerase Chain Reaction)

←Older revision		Revision as of 20:32, 16 July 2007
Line 74:		Line 74:
	*dNTPs (ATGC)		*dNTPs (ATGC)
	*Heat-stable DNA polymerase<br>		*Heat-stable DNA polymerase<br>
		+
		+
	A PCR cycle consists of three steps.		A PCR cycle consists of three steps.
	*Strand separation. The two strands of the parent DNA molecule are separated by heating the solution to 95ºC.		*Strand separation. The two strands of the parent DNA molecule are separated by heating the solution to 95ºC.
	*Hybridization of primers. The solution is cooled to 54ºC to allow each primer to bind to a DNA strand. One primer hybridizes to the 3’end of the target on one strand, and the other primer hybridizes to the 3’ end on the complementary target strand.		*Hybridization of primers. The solution is cooled to 54ºC to allow each primer to bind to a DNA strand. One primer hybridizes to the 3’end of the target on one strand, and the other primer hybridizes to the 3’ end on the complementary target strand.
	*DNA synthesis. The solution is heated to 72ºC, the optimal temperature for Taq DNA polymerase (a heat-stable DNA polymerase from a thermophilic bacterium). The polymerase elongates both primers in the direction of the target sequence because DNA synthesis is in the 5’-to-3’ direction.<br>		*DNA synthesis. The solution is heated to 72ºC, the optimal temperature for Taq DNA polymerase (a heat-stable DNA polymerase from a thermophilic bacterium). The polymerase elongates both primers in the direction of the target sequence because DNA synthesis is in the 5’-to-3’ direction.<br>
-	These three steps constitute one cycle of the PCR amplification. The duplexes are heated to begin the second cycle, which produces four duplexes, and then the third cycle is initiated. After n cycles, the sequence is amplified 2n-fold. There are several noteworthy features of this method. First, the sequence of the target need not be known. Only knowledge of the flanking sequences is required. Second, the target can be much larger than the primers. (>10 kb) Third, primers do not have to perfectly match flanking sequences. Primers derived from a gene of known sequence can be used to amplify related genes of the same family. Fourth, stringency or the closeness of the match between primer and target can be controlled by temperature and salt (MgCl2). Fifth, PCR is very sensitive. A single DNA molecule can be amplified and detected.	+	These three steps constitute one cycle of the PCR amplification. The duplexes are heated to begin the second cycle, which produces four duplexes, and then the third cycle is initiated. After n cycles, the sequence is amplified 2n-fold.
		+
		+
		+	There are several noteworthy features of this method. First, the sequence of the target need not be known. Only knowledge of the flanking sequences is required. Second, the target can be much larger than the primers. (>10 kb) Third, primers do not have to perfectly match flanking sequences. Primers derived from a gene of known sequence can be used to amplify related genes of the same family. Fourth, stringency or the closeness of the match between primer and target can be controlled by temperature and salt (MgCl2). Fifth, PCR is very sensitive. A single DNA molecule can be amplified and detected.

	== DNA Sequencing ==		== DNA Sequencing ==

Cheuk Ka Tong: /* Recombinant DNA */

Cheuk Ka Tong — Mon, 16 Jul 2007 20:29:54 GMT

Recombinant DNA

←Older revision		Revision as of 20:29, 16 July 2007
Line 28:		Line 28:
	[[IMAGE:Recombinant_DNA.jpg\|frame\|Inserting DNA of interest into the plasmid]]		[[IMAGE:Recombinant_DNA.jpg\|frame\|Inserting DNA of interest into the plasmid]]

-	Recombinant DNA is the construction of new combinations of unrelated genes. These novel combinations can be cloned and amplified by introducing them into host cells where they are replicated by the DNA-synthesizing machinery of the host. A DNA fragment of interest is covalently joined to a DNA vector. A vector can replicate autonomously in an appropriate host. Plasmids (naturally occurring circles of DNA in bacteria) and bacteriophage λ (a virus) are common vectors for cloning in E.coli. The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.	+	Recombinant DNA is the construction of new combinations of unrelated genes. These novel combinations can be cloned and amplified by introducing them into host cells where they are replicated by the DNA-synthesizing machinery of the host. A DNA fragment of interest is covalently joined to a DNA vector. A vector can replicate autonomously in an appropriate host. Plasmids (naturally occurring circles of DNA in bacteria) and bacteriophage λ (a virus) are common vectors for cloning in E.coli.
		+
		+
		+	The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.

	== Gene Cloning ==		== Gene Cloning ==

Cheuk Ka Tong: /* Gene Cloning */

Cheuk Ka Tong — Mon, 16 Jul 2007 20:29:18 GMT

Gene Cloning

←Older revision		Revision as of 20:29, 16 July 2007
Line 31:		Line 31:

	== Gene Cloning ==		== Gene Cloning ==
-	One of the most useful plasmids for cloning is pBR322. pBR322 contains genes for resistance to tetracycline and ampicillin (antibiotics like penicillin). Different endonucleases can cleave this plasmid at a variety of unique sites, at which DNA fragments can be inserted. Insertion of DNA at the EcoRI restriction site does not alter either of the genes for antibiotic resistance. However insertion at the SalI or PstI site inactivates the gene for tetracycline or ampicillin resistance, an effect called insertional inactivation. Cells containing pBR322 with a DNA insert at the SalI site are resistant to ampicillin but sensitive to tetracycline, so they can be readily selected. Cells that failed to take up the vector are sensitive to both antibiotics. Cells that take up pBR322 without a DNA insert are resistant to both. The colony of cells that are shown to have taken up the correct recombinant DNA is grown to produce a large amount of the DNA of interest.	+
		+	[[IMAGE:Plasmid_pBR322.jpg\|frame\|Plasmid pBR322]]
		+
		+	One of the most useful plasmids for cloning is pBR322. pBR322 contains genes for resistance to tetracycline and ampicillin (antibiotics like penicillin). Different endonucleases can cleave this plasmid at a variety of unique sites, at which DNA fragments can be inserted. Insertion of DNA at the EcoRI restriction site does not alter either of the genes for antibiotic resistance. However insertion at the SalI or PstI site inactivates the gene for tetracycline or ampicillin resistance, an effect called insertional inactivation.
		+
		+
		+	Cells containing pBR322 with a DNA insert at the SalI site are resistant to ampicillin but sensitive to tetracycline, so they can be readily selected. Cells that failed to take up the vector are sensitive to both antibiotics. Cells that take up pBR322 without a DNA insert are resistant to both. The colony of cells that are shown to have taken up the correct recombinant DNA is grown to produce a large amount of the DNA of interest.

	== Gel Electrophoresis ==		== Gel Electrophoresis ==

Cheuk Ka Tong: /* Gene Cloning */

Cheuk Ka Tong — Mon, 16 Jul 2007 20:22:06 GMT

Gene Cloning

←Older revision		Revision as of 20:22, 16 July 2007
Line 31:		Line 31:

	== Gene Cloning ==		== Gene Cloning ==
		+	One of the most useful plasmids for cloning is pBR322. pBR322 contains genes for resistance to tetracycline and ampicillin (antibiotics like penicillin). Different endonucleases can cleave this plasmid at a variety of unique sites, at which DNA fragments can be inserted. Insertion of DNA at the EcoRI restriction site does not alter either of the genes for antibiotic resistance. However insertion at the SalI or PstI site inactivates the gene for tetracycline or ampicillin resistance, an effect called insertional inactivation. Cells containing pBR322 with a DNA insert at the SalI site are resistant to ampicillin but sensitive to tetracycline, so they can be readily selected. Cells that failed to take up the vector are sensitive to both antibiotics. Cells that take up pBR322 without a DNA insert are resistant to both. The colony of cells that are shown to have taken up the correct recombinant DNA is grown to produce a large amount of the DNA of interest.

	== Gel Electrophoresis ==		== Gel Electrophoresis ==

Cheuk Ka Tong: /* Recombinant DNA */

Cheuk Ka Tong — Mon, 16 Jul 2007 20:19:49 GMT

Recombinant DNA

←Older revision		Revision as of 20:19, 16 July 2007
Line 25:		Line 25:

	== Recombinant DNA ==		== Recombinant DNA ==
		+
		+	[[IMAGE:Recombinant_DNA.jpg\|frame\|Inserting DNA of interest into the plasmid]]
		+
	Recombinant DNA is the construction of new combinations of unrelated genes. These novel combinations can be cloned and amplified by introducing them into host cells where they are replicated by the DNA-synthesizing machinery of the host. A DNA fragment of interest is covalently joined to a DNA vector. A vector can replicate autonomously in an appropriate host. Plasmids (naturally occurring circles of DNA in bacteria) and bacteriophage λ (a virus) are common vectors for cloning in E.coli. The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.		Recombinant DNA is the construction of new combinations of unrelated genes. These novel combinations can be cloned and amplified by introducing them into host cells where they are replicated by the DNA-synthesizing machinery of the host. A DNA fragment of interest is covalently joined to a DNA vector. A vector can replicate autonomously in an appropriate host. Plasmids (naturally occurring circles of DNA in bacteria) and bacteriophage λ (a virus) are common vectors for cloning in E.coli. The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.

Cheuk Ka Tong: /* Recombinant DNA */

Cheuk Ka Tong — Mon, 16 Jul 2007 20:16:22 GMT

Recombinant DNA

←Older revision		Revision as of 20:16, 16 July 2007
Line 25:		Line 25:

	== Recombinant DNA ==		== Recombinant DNA ==
		+	Recombinant DNA is the construction of new combinations of unrelated genes. These novel combinations can be cloned and amplified by introducing them into host cells where they are replicated by the DNA-synthesizing machinery of the host. A DNA fragment of interest is covalently joined to a DNA vector. A vector can replicate autonomously in an appropriate host. Plasmids (naturally occurring circles of DNA in bacteria) and bacteriophage λ (a virus) are common vectors for cloning in E.coli. The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. The DNA fragment of interest is prepared from a larger piece of DNA using the same restriction enzyme as was used to open the plasmid DNA. The single-stranded ends of the fragment are then complementary to those of the cut plasmid. The DNA fragment and the cut plasmid are annealed and then joined by DNA ligase. DNA ligase catalyzes the formation of a phosphodiester bond at a break in a DNA chain. An energy source such as ATP is required for the joining reaction.
		+
	== Gene Cloning ==		== Gene Cloning ==