Endy:Double stranding oligo libraries - Revision history

Kchang17: /* Perform PCR cleanup on the double-stranded library */

2006-08-29T19:39:51Z

Perform PCR cleanup on the double-stranded library

←Older revision		Revision as of 19:39, 29 August 2006
Line 39:		Line 39:
	** 100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column		** 100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column
	* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)		* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
-	* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)	+	* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded) [[Image:Double-stranded_oligo_libraries.jpg\|thumb\|none\|300px\|Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)]]
-	** [[Image:Double-stranded_oligo_libraries.jpg\|thumb\|none\|~~250px~~\|Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)]]	+

	==[[Restriction digest]] the library==		==[[Restriction digest]] the library==

Kchang17: /* Perform PCR cleanup on the double-stranded library */

2006-08-29T19:38:41Z

Perform PCR cleanup on the double-stranded library

←Older revision		Revision as of 19:38, 29 August 2006
Line 40:		Line 40:
	* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)		* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
	* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)		* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)
-	** [[Image:Double-stranded_oligo_libraries.jpg\|thumb\|~~left~~\|250px\|Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)]]	+	** [[Image:Double-stranded_oligo_libraries.jpg\|thumb\|none\|250px\|Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)]]

	==[[Restriction digest]] the library==		==[[Restriction digest]] the library==

Kchang17: /* Perform PCR cleanup on the double-stranded library */

2006-08-29T19:38:23Z

Perform PCR cleanup on the double-stranded library

←Older revision		Revision as of 19:38, 29 August 2006
Line 40:		Line 40:
	* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)		* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
	* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)		* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)
		+	** [[Image:Double-stranded_oligo_libraries.jpg\|thumb\|left\|250px\|Three libraries ~100bp; on the left is the single-stranded oligo; on the right are double-stranded oligos (different lanes are different primers)]]

	==[[Restriction digest]] the library==		==[[Restriction digest]] the library==

Kchang17: /* Perform PCR cleanup on the double-stranded library */

2006-08-28T21:46:09Z

Perform PCR cleanup on the double-stranded library

←Older revision		Revision as of 21:46, 28 August 2006
Line 36:		Line 36:
	==Perform PCR cleanup on the double-stranded library==		==Perform PCR cleanup on the double-stranded library==
	* This concentrates the samples and allows for the buffer to be switched to something more appropriate.		* This concentrates the samples and allows for the buffer to be switched to something more appropriate.
-	* PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column)	+	* PCR purification columns can handle up to 10ug of DNA
		+	** 100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column
	* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)		* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
	* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)		* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)

Kchang17 at 21:45, 28 August 2006

2006-08-28T21:45:44Z

←Older revision		Revision as of 21:45, 28 August 2006
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	*4<sup>o</sup>C forever		*4<sup>o</sup>C forever

-	==PCR cleanup on the double-stranded ~~libraries~~==	+	==Perform PCR cleanup on the double-stranded library==
	* This concentrates the samples and allows for the buffer to be switched to something more appropriate.		* This concentrates the samples and allows for the buffer to be switched to something more appropriate.
	* PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column)		* PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column)
	* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)		* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)
		+	* You can run a sample of the PCR product out on a gel against a sample of the original library to verify that the double stranding worked (double stranded DNA should run slightly faster than single stranded)

-	==[[Restriction digest]] the ~~libraries~~==	+	==[[Restriction digest]] the library==

-	==~~Separate on a [[Agarose_gel_electrophoresis\|gel]] and do a second~~ PCR cleanup==	+	==Perform PCR cleanup on the digest==
-	* Alternatively, you can run a sample of the first PCR reaction out on a gel for analysis against a sample of the original library (double stranded should run slightly faster than single stranded), then perform the digest. Doing a PCR cleanup on the digest will remove the cut ends, since they are small.	+	* This will remove the cut ends, since they are small.

	==[[DNA_Ligation\|Ligate]] the sample from the PCR cleanup with a vector==		==[[DNA_Ligation\|Ligate]] the sample from the PCR cleanup with a vector==

Kchang17: /* PCR cleanup on the double-stranded libraries */

2006-08-28T21:40:06Z

PCR cleanup on the double-stranded libraries

←Older revision		Revision as of 21:40, 28 August 2006
Line 36:		Line 36:
	==PCR cleanup on the double-stranded libraries==		==PCR cleanup on the double-stranded libraries==
	* This concentrates the samples and allows for the buffer to be switched to something more appropriate.		* This concentrates the samples and allows for the buffer to be switched to something more appropriate.
-	* PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug)	+	* PCR purification columns can handle up to 10ug of DNA (100pmol of a 100bp oligo is about 3ug, so multiple 100-ul reactions of 25pmol can be combined into one column)
	* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)		* Expected recovery from a PCR purification reaction is 90% (from the Invitrogen package)

Kchang17: /* PCR protocol */

2006-08-28T21:38:39Z

PCR protocol

←Older revision		Revision as of 21:38, 28 August 2006
Line 27:		Line 27:

	===PCR protocol===		===PCR protocol===
-	*95 <sup>o</sup>C for 2.5 minutes	+	*95<sup>o</sup>C for 2.5 minutes
	*Cycle 5 times:		*Cycle 5 times:
-	**55 <sup>o</sup>C (or whatever temperature is appropriate) for 30 seconds (annealing)	+	**55<sup>o</sup>C (or whatever temperature is appropriate) for 30 seconds (annealing)
-	**72 <sup>o</sup>C for 1.5 minutes (elongation)	+	**72<sup>o</sup>C for 1.5 minutes (elongation)
-	*72 <sup>o</sup>C for 10 minutes (final elongation)	+	*72<sup>o</sup>C for 10 minutes (final elongation)
-	*4 <sup>o</sup>C forever	+	*4<sup>o</sup>C forever

	==PCR cleanup on the double-stranded libraries==		==PCR cleanup on the double-stranded libraries==

Kchang17: /* PCR protocol */

2006-08-28T21:38:12Z

PCR protocol

←Older revision		Revision as of 21:38, 28 August 2006
Line 27:		Line 27:

	===PCR protocol===		===PCR protocol===
-	*95 C for 2.5 minutes	+	*95 <sup>o</sup>C for 2.5 minutes
	*Cycle 5 times:		*Cycle 5 times:
-	**55 C (or whatever temperature is appropriate) for 30 s (annealing)	+	**55 <sup>o</sup>C (or whatever temperature is appropriate) for 30 seconds (annealing)
-	**72 C for 1.5 minutes (elongation)	+	**72 <sup>o</sup>C for 1.5 minutes (elongation)
-	*72 C for 10 minutes (final elongation)	+	*72 <sup>o</sup>C for 10 minutes (final elongation)
-	*4 C forever	+	*4 <sup>o</sup>C forever

	==PCR cleanup on the double-stranded libraries==		==PCR cleanup on the double-stranded libraries==

Kchang17: /* Double strand the library with modified PCR */

2006-08-28T21:36:15Z

Double strand the library with modified PCR

←Older revision		Revision as of 21:36, 28 August 2006
Line 12:		Line 12:
	==Double strand the library with modified PCR==		==Double strand the library with modified PCR==
	*Expected max library size is 10<sup>8</sup> molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 10<sup>9</sup> molecules for a single transformation.		*Expected max library size is 10<sup>8</sup> molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 10<sup>9</sup> molecules for a single transformation.
-	**~~1 pmol~~ corresponds to ~10<sup>11</sup> molecules	+	**1pmol corresponds to ~10<sup>11</sup> molecules
	**Use 25pmol of library to make enough for 2500 transformations		**Use 25pmol of library to make enough for 2500 transformations
	*Total library DNA should be less than ~25pmol per 100uL reaction		*Total library DNA should be less than ~25pmol per 100uL reaction

Kchang17: /* Double strand the library with modified PCR */

2006-08-28T21:34:39Z

Double strand the library with modified PCR

←Older revision		Revision as of 21:34, 28 August 2006
Line 11:		Line 11:

	==Double strand the library with modified PCR==		==Double strand the library with modified PCR==
-	* ~~Total~~ library ~~DNA should be~~ <~~25pmol per 100uL reaction~~	+	*Expected max library size is 10<sup>8</sup> molecules (limit set by transformation efficiency.) You want to load 10X the expected library size for a single library construction. Therefore, you would like to have 10<sup>9</sup> molecules for a single transformation.
-	* You want to ~~start with~~ 10X the ~~final desired amount of~~ library for ~~PCR~~	+	**1 pmol corresponds to ~10<sup>11</sup> molecules
-	** ~~Split into separate~~ 100uL ~~reactions if necessary~~	+	**Use 25pmol of library to make enough for 2500 transformations
		+	*Total library DNA should be less than ~25pmol per 100uL reaction

-	===Reaction Mix (100uL)===	+	===Reaction Mix (100uL, 25pmol library)===
	Use the following reaction mix for each PCR reaction:		Use the following reaction mix for each PCR reaction:
	*10 μl 10x Thermo polymerase buffer		*10 μl 10x Thermo polymerase buffer