Addition of 3' A overhangs to PCR products - Revision history

Torsten Waldminghaus: add back to protocol link

2009-02-23T16:33:18Z

add back to protocol link

←Older revision		Revision as of 16:33, 23 February 2009
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		+	{{back to protocols}}
		+
	==Curators==		==Curators==
	[[User:S. Mohsen Hosseini\|Mohsen Hosseini]]		[[User:S. Mohsen Hosseini\|Mohsen Hosseini]]

Torsten Waldminghaus: /* Curators */

2009-02-18T22:18:05Z

Curators

←Older revision		Revision as of 22:18, 18 February 2009
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	==Curators==		==Curators==
-	Mohsen Hosseini	+	[[User:S. Mohsen Hosseini\|Mohsen Hosseini]]

	==Abstract==		==Abstract==

Torsten Waldminghaus at 22:16, 18 February 2009

2009-02-18T22:16:40Z

←Older revision		Revision as of 22:16, 18 February 2009
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	Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.		Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.

-	[[Category:Protocol]]	+	[[Category:Protocol]][[Category:Needs attention]]

Torsten Waldminghaus: /* Abstract */

2009-02-18T22:13:38Z

Abstract

←Older revision		Revision as of 22:13, 18 February 2009
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	==Abstract==		==Abstract==
-	If PCR is performed using a proofreading DNA polymerase, such as ''Pfu'' DNA polymerase, the product will have blunt ends. ''Taq'' DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.	+	If PCR is performed using a proofreading DNA polymerase, such as ''[[Pfu]]'' DNA polymerase, the product will have blunt ends. ''Taq'' DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.

	==Materials==		==Materials==

S. Mohsen Hosseini: /* Critical steps */

2008-10-19T04:00:54Z

Critical steps

←Older revision		Revision as of 04:00, 19 October 2008
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	==Critical steps==		==Critical steps==
-		+	Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation) ; since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.

	==Troubleshooting==		==Troubleshooting==

S. Mohsen Hosseini: /* Procedure */

2008-10-19T04:00:42Z

Procedure

←Older revision		Revision as of 04:00, 19 October 2008
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	==Procedure==		==Procedure==
	1. Purify the PCR product [[Image:Critical step.png]]		1. Purify the PCR product [[Image:Critical step.png]]
-	Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation) ; since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.	+
-		+
	2. Prepare Taq DNA polymerase reaction mix for a typical 20 - 50 μl reaction:		2. Prepare Taq DNA polymerase reaction mix for a typical 20 - 50 μl reaction:

S. Mohsen Hosseini: /* Procedure */

2008-10-19T03:59:26Z

Procedure

←Older revision		Revision as of 03:59, 19 October 2008
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	==Procedure==		==Procedure==
-	1. Purify the PCR product	+	1. Purify the PCR product [[Image:Critical step.png]]
	Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation) ; since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.		Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation) ; since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.

S. Mohsen Hosseini: /* Procedure */

2008-10-19T03:57:12Z

Procedure

←Older revision		Revision as of 03:57, 19 October 2008
Line 18:		Line 18:

	==Procedure==		==Procedure==
		+	1. Purify the PCR product
		+	Before adding the overhangs it is very important to remove all the Proofreading DNA Polymerase (Pfu) by purifying the PCR product carefully (e.g. with a commercial PCR purification kit or phenol extraction and DNA precipitation) ; since the proofreading activity of DNA Polymerase will degrade the A overhangs, creating blunt ends again.

		+	2. Prepare Taq DNA polymerase reaction mix for a typical 20 - 50 μl reaction:
		+
		+	{\| class="wikitable" style="text-align:center"; border="1"
		+	! !! Final Concentration!! Volume(μl)
		+	\|-
		+	\| Purified PCR product \|\| 0.15 to 1.5 pmol\|\| variable
		+	\|-
		+	\| dATP (10 mM)\|\| 0.2 mM \|\| 1
		+	\|-
		+	\| PCR Buffer with Mg (10x) \|\| 1x (1.5 mM MgCl<sub>2</sub>)\|\| 5
		+	\|-
		+	\| Taq DNA Polymerase (5 U/μl) \|\| 1U \|\| 0.2
		+	\|-
		+	\| ddH<sub>2</sub>O \|\| - \|\| up to 50 μl
		+	\|}
		+
		+	The A-addition reaction works best when a specific amount of the PCR product is used. The recommended amount is 10–100 ng PCR product for each 100 bp length of the PCR product. This corresponds to 0.15–1.5 pmol PCR product (see table below).
		+
		+	{\| class="wikitable" style="text-align:center"; border="1"
		+	! PCR product size !! Amount of PCR product to use
		+	\|-
		+	\| 100 bp \|\| 10–100 ng
		+	\|-
		+	\| 250 bp \|\| 25–250 ng
		+	\|-
		+	\| 1000 bp \|\| 100–1000 ng
		+	\|}
		+
		+	3. Incubate 20 min at 72 °C.
		+
		+	4. Proceed to TA cloning. For optimal ligation efficiency, it's best to use fresh PCR products, since 3´A-overhangs will gradually be lost during storage.

	==Critical steps==		==Critical steps==

S. Mohsen Hosseini: /* Materials */

2008-10-19T03:41:58Z

Materials

←Older revision		Revision as of 03:41, 19 October 2008
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	===Reagents===		===Reagents===
-		+	*Purified PCR product
		+	*dATP
		+	*PCR buffer with Mg
		+	*''Taq'' DNA Polymerase
		+	*ddH<sub>2</sub>O
	===Equipment===		===Equipment===
		+	*Thermocycler or Heat block equilibrated at 72°C
		+	*Spectrophotometer

	==Procedure==		==Procedure==

S. Mohsen Hosseini: /* Abstract */

2008-10-19T03:35:07Z

Abstract

←Older revision		Revision as of 03:35, 19 October 2008
Line 3:		Line 3:

	==Abstract==		==Abstract==
		+	If PCR is performed using a proofreading DNA polymerase, such as ''Pfu'' DNA polymerase, the product will have blunt ends. ''Taq'' DNA polymerase catalyzes the non-template directed addition of an adenine residue to the 3´-end of both strands of DNA molecules to make it suitable for TA cloning.

	==Materials==		==Materials==