Genomic Insertion Protocol - Revision history

Wideloache at 15:42, 15 May 2009

2009-05-15T15:42:16Z

Wideloache at 14:10, 15 May 2009

2009-05-15T14:10:22Z

Wideloache at 14:01, 15 May 2009

2009-05-15T14:01:28Z

Wideloache at 13:53, 15 May 2009

2009-05-15T13:53:46Z

Wideloache at 13:47, 15 May 2009

2009-05-15T13:47:29Z

Wideloache at 13:46, 15 May 2009

2009-05-15T13:46:50Z

Wideloache at 02:33, 16 March 2009

2009-03-16T02:33:25Z

Wideloache at 02:33, 16 March 2009

2009-03-16T02:33:09Z

Wideloache at 02:32, 16 March 2009

2009-03-16T02:32:50Z

Wideloache at 10:29, 17 December 2008

2008-12-17T10:29:27Z

@@ Line 26: / Line 26: @@
 #Confirm using primers attPhi80-1 and attPhi80-4 (sequence given below - oligos already purchased in the Campbell Lab) to sequence the genomic DNA.
-You can also perform PCR on the integrant colonies using the 4 primers shown below. attPhi80-1 and attPhi80-4 bind to the phi80 site in the genome. attPhi80-2 and attPhi80-3 bind to the attB site on the pG80ko plasmid. The expected results are given in the CRIM paper, and shown in the table below. I found that this procedure gave some unexpected results for my controls and would recommend sequencing instead of PCR for confirmation of the integrants.
+You can also perform PCR on the integrant colonies using the 4 primers shown below. attPhi80-1 and attPhi80-4 bind to the phi80 site in the genome. attPhi80-2 and attPhi80-3 bind to the attB site on the pG80ko plasmid. The expected results are given in the CRIM paper, and shown in the table below. I found that this procedure gave some unexpected results (see gel below) for my controls and would recommend sequencing instead of PCR for confirmation of the integrants.
 attPhi80-1: CTGCTTGTGGTGGTGAAT
@@ Line 42: / Line 42: @@
 |63||546||409, 732||409, 595, 732
 |}

@@ Line 17: / Line 17: @@
 #Plate the transformation on gentamicin plates at 15ug/ml
 #Grown up a colony from the transformation, miniprep it, and perform a digestion to verify successful ligation. Save the miniprep!
-#Y
+You can also perform PCR on the integrant colonies using the 4 primers shown below. attPhi80-1 and attPhi80-4 bind to the phi80 site in the genome. attPhi80-2 and attPhi80-3 bind to the attB site on the pG80ko plasmid. The expected results are given in the CRIM paper, and shown in the table below. I found that this procedure gave some unexpected results for my controls and would recommend sequencing instead of PCR for confirmation of the integrants.
 attPhi80-1: CTGCTTGTGGTGGTGAAT

@@ Line 8: / Line 8: @@
 pG80ko contains the R6K origin of replication, which is only
-active in the presence of Pir protein. This plasmid will eventually be inserted entirely into the genome of E. coli. The helper plasmid, pInt80-649, can only be replicated inside E. coli l at temperatures below 43�C because the CI857 protein, which is necessary for replication, is inactivated at high temperatures. In order to perform a genomic insertion, the DNA to be inserted is placed on pG80ko using an EcoRI/PstI digestion. This plasmid is transformed into cells that already contain pInt80-649 (and thus express Pir). PG80ko carries an �80 attP site that allows for recombination with the �80 attB site in the E. coli genome. This recombination event is aided by the integrase which is expressed by the helper plasmid, pInt80-649. Cells that have undergone a recombination event can be selected by growth on gentamicin plates at 43�C. High temperatures cause the cells to stop replication of the helper plasmid and consequently stop production of Pir too. Without Pir, pG80ko is incapable of replication unless the plasmid has been inserted into the genome.
+active in the presence of Pir protein. This plasmid will eventually be inserted entirely into the genome of E. coli. The helper plasmid, pInt80-649, can only be replicated inside E. coli l at temperatures below 43C because the CI857 protein, which is necessary for replication, is inactivated at high temperatures. In order to perform a genomic insertion, the DNA to be inserted is placed on pG80ko using an EcoRI/PstI digestion. This plasmid is transformed into cells that already contain pInt80-649 (and thus express Pir). PG80ko carries an phi80 attP site that allows for recombination with the phi80 attB site in the E. coli genome. This recombination event is aided by the integrase which is expressed by the helper plasmid, pInt80-649. Cells that have undergone a recombination event can be selected by growth on gentamicin plates at 43C. High temperatures cause the cells to stop replication of the helper plasmid and consequently stop production of Pir too. Without Pir, pG80ko is incapable of replication unless the plasmid has been inserted into the genome.
-With that background, the steps for performing this insertion will be listed below:
+With that background, the steps for performing this insertion are listed below:
-#Test
+#Perform an EcoRI/PstI digestion on the pG80ko plasmid with a stuffer insert and gel purify the vector. Also perform an EcoRI/PstI digestion and gel purification of the part to be inserted. This part is probably contained in an BioBrick standard vector.
 Note that you'll need a pir strain for replication of pG80 plasmids.  You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells.  Grow up, miniprep, and map a single colony.  Make competent cells of pInt80-649 in your target strain (plate them on Amp).  It is temperature sensitive, so do all growth manipulations at 30 degrees.  Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees.  Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees.  You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

@@ Line 7: / Line 7: @@
 [[Image:PG80ko.jpg|400px|thumb|none|pG80ko containing BBa_K091206 for insertion. This is where your part will be.]] [[Image:PInt80.png|400px|thumb|none|pInt80-649.]]
+#Test
 Note that you'll need a pir strain for replication of pG80 plasmids.  You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells.  Grow up, miniprep, and map a single colony.  Make competent cells of pInt80-649 in your target strain (plate them on Amp).  It is temperature sensitive, so do all growth manipulations at 30 degrees.  Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees.  Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees.  You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

@@ Line 1: / Line 1: @@
 This genomic insertion protocol utilizes [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=100134 CRIM technology] and was modified by the [http://andersonlab.qb3.berkeley.edu/ Anderson Lab at UC Berkeley]. For more information on the technology, you can read the [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=100134 article on CRIM technology].
@@ Line 8: / Line 5: @@
 This plasmid maps are shown below:
-[[Image:PG80ko.jpg|200px|thumb|none|pG80ko containing BBa_K091206 for insertion. This is where your part will be.]] [[Image:PInt80.png|200px|thumb|none|pInt80-649.]]
+[[Image:PG80ko.jpg|400px|thumb|none|pG80ko containing BBa_K091206 for insertion. This is where your part will be.]] [[Image:PInt80.png|400px|thumb|none|pInt80-649.]]

← Older revision		Revision as of 13:46, 15 May 2009
Line 1:		Line 1:
	Warning: This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my [http://gcat.davidson.edu/GcatWiki/index.php/Will_DeLoache_Notebook lab notebook results] until then.**		Warning: This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my [http://gcat.davidson.edu/GcatWiki/index.php/Will_DeLoache_Notebook lab notebook results] until then.**
		+
		+
		+	This genomic insertion protocol utilizes [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=100134 CRIM technology] and was modified by the [http://andersonlab.qb3.berkeley.edu/ Anderson Lab at UC Berkeley]. For more information on the technology, you can read the [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=100134 article on CRIM technology].
		+
		+	You will need two plasmids: pG80ko and pInt80-649. pG80ko can be found in the [http://gcat.davidson.edu/GCATalog-r1/Login.php freezer stocks] in box 4-1, position 55. It contains a stuffer insert that is about 700 bps and should be replaced with the part you want to insert. pInt80-649 can be found in the freezer stocks in box 4-1, position 51. pG80ko's sequence can be found [http://gcat.davidson.edu/GcatWiki/images/b/bb/PG80ko.doc here]. pIn80-649's sequence can be found [http://gcat.davidson.edu/GcatWiki/images/e/ec/PInt80-649.doc here].
		+
		+	This plasmid maps are shown below:
		+
		+	[[Image:PG80ko.jpg\|200px\|thumb\|none\|pG80ko containing BBa_K091206 for insertion. This is where your part will be.]] [[Image:PInt80.png\|200px\|thumb\|none\|pInt80-649.]]
		+
		+

← Older revision		Revision as of 02:33, 16 March 2009
Line 1:		Line 1:
	Warning: This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my [http://gcat.davidson.edu/GcatWiki/index.php/Will_DeLoache_Notebook lab notebook results] until then.**		Warning: This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my [http://gcat.davidson.edu/GcatWiki/index.php/Will_DeLoache_Notebook lab notebook results] until then.**
		+
		+

	Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.		Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

← Older revision		Revision as of 02:32, 16 March 2009
Line 1:		Line 1:
		+	*This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my [http://gcat.davidson.edu/GcatWiki/index.php/Will_DeLoache_Notebook lab notebook results] until then.*
		+
	Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.		Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

← Older revision		Revision as of 10:29, 17 December 2008
Line 1:		Line 1:
	Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.		Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.
		+
		+	attPhi80-1: CTGCTTGTGGTGGTGAAT
		+
		+	attPhi80-2: ACTTAACGGCTGACATGG
		+
		+	attPhi80-3: ACGAGTATCGAGATGGCA
		+
		+	attPhi80-4: TAAGGCAAGACGATCAGG

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