Seth:Quick Transformant Screen - Revision history

Reshma P. Shetty at 01:33, 10 May 2007

Reshma P. Shetty — Thu, 10 May 2007 01:33:54 GMT

←Older revision		Revision as of 01:33, 10 May 2007
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	*[[User:Seth]]		*[[User:Seth]]

-	[[Category:Protocol]]	+	[[Category:Protocol]] [[Category:In vitro]] [[Category:Escherichia coli]] [[Category:DNA]]

Btarlow: /* Notes */

Btarlow — Wed, 21 Mar 2007 19:39:11 GMT

Notes

←Older revision		Revision as of 19:39, 21 March 2007
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	*Btarlow: note		*Btarlow: note
-	I do this with a couple modifications. #I resuspend my colony directly in 10ul LB broth. This way, I don't worry about lysis. Sterile LB won't affect the PCR, but it's always good to include a negative control. #Then I use 1uL of the culture as a template for 50uL PCR reaction. #When avaiable, I like to use primers to the vector that span the multiple cloning site (MCS) so that I always get PCR product for every template. Many plasmids already have sequences designed for sequencing primers (ie T7, T3, M13F, M13R) and these work well. In a recent experiment, I ligated a 1800bp fragment into the backbone. If I had empty vector, I saw a 250bp band. If the plasmid contained the insert, I got a 2050bp band.	+	I do this with a couple modifications.
		+	#I resuspend my colony directly in 10ul LB broth. This way, I don't worry about lysis. Sterile LB won't affect the PCR, but it's always good to include a negative control.
		+	#Then I use 1uL of the culture as a template for 50uL PCR reaction.
		+	#When avaiable, I like to use primers to the vector that span the multiple cloning site (MCS) so that I always get PCR product for every template. Many plasmids already have sequences designed for sequencing primers built in (ie T7, T4, M13F, M13R) and these work well. In a recent experiment, I ligated a 1800bp fragment into the backbone. If I had empty vector, I saw a 250bp band. If the plasmid contained the insert, I got a 2050bp band.

	==Contact==		==Contact==

Btarlow: /* Notes */

Btarlow — Wed, 21 Mar 2007 19:37:48 GMT

Notes

←Older revision		Revision as of 19:37, 21 March 2007
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	==Notes==		==Notes==
	*This protocol is meant to be a time and resource saving method, but is not appropriate for every ligation and		*This protocol is meant to be a time and resource saving method, but is not appropriate for every ligation and
-
	transformation. Experiments that yield a large range of fragment sizes or very large fragments, such as BAC or genomic		transformation. Experiments that yield a large range of fragment sizes or very large fragments, such as BAC or genomic
		+	digests (shotgun type cloning), may exceed the limitations of PCR or your particular ''taq''. Very large inserts may necessitate such lengthy extension times that the PCR is no longer a time saving tool. Still, with a pool of fragments we often find this technique useful as it lets us select one or two clones of each size, or choose the clone with our expected length product.

-	~~digests (shotgun type cloning), may exceed the limitations of PCR or your particular ''taq''. Very large inserts may~~	+	*Because you are priming your PCR off the plasmid, there will be a small fragment in every lane of your gel, even if a clone had no insert. This also means that when looking on your gel you want to factor in that additional DNA when approximating fragment length. If a lane is completely empty there may have been a problem with your PCR. A template negative PCR reaction is always a recommended control.
-		+
-	~~necessitate such lengthy extension times that the PCR is no longer a time saving tool. Still, with a pool of fragments we~~	+
-		+
-	~~often find this technique useful as it lets us select one or two clones of each size, or choose the clone with our expected~~	+
-		+
-	~~length product.~~	+
-		+
-	*Because you are priming your PCR off the plasmid, there will be a small fragment in every lane of your gel, even if a clone	+
-		+
-	had no insert. This also means that when looking on your gel you want to factor in that additional DNA when approximating	+
-		+
-	fragment length. If a lane is completely empty there may have been a problem with your PCR. A template negative PCR reaction	+

-	is always a ~~recommended~~ control.	+	*Btarlow: note
		+	I do this with a couple modifications. #I resuspend my colony directly in 10ul LB broth. This way, I don't worry about lysis. Sterile LB won't affect the PCR, but it's always good to include a negative control. #Then I use 1uL of the culture as a template for 50uL PCR reaction. #When avaiable, I like to use primers to the vector that span the multiple cloning site (MCS) so that I always get PCR product for every template. Many plasmids already have sequences designed for sequencing primers (ie T7, T3, M13F, M13R) and these work well. In a recent experiment, I ligated a 1800bp fragment into the backbone. If I had empty vector, I saw a 250bp band. If the plasmid contained the insert, I got a 2050bp band.

	==Contact==		==Contact==

Btarlow: /* Procedure */

Btarlow — Wed, 21 Mar 2007 19:30:21 GMT

Procedure

←Older revision		Revision as of 19:30, 21 March 2007
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	#Grow plates overnight at 37°C		#Grow plates overnight at 37°C
	#The next day prepare a 96-well plate by adding 20 μL of MilliQ water to as many wells as you have colonies for.		#The next day prepare a 96-well plate by adding 20 μL of MilliQ water to as many wells as you have colonies for.
-	#Pick a colony and resuspend thoroughly in the water by pipetting. Cells will lyse over the next several minutes. Begin setting up your PCR immediately.	+	#Pick a colony with a micropipet tip and resuspend thoroughly in the water by pipetting. Cells will lyse over the next several minutes. Begin setting up your PCR immediately. Alternatively, bacteria could be resuspended in LB without risk of lysis. Include a negative control.
	#Prepare a master mix for PCR.		#Prepare a master mix for PCR.
	#Use 10μL of your lysed cells for template.		#Use 10μL of your lysed cells for template.

Seth Kasowitz: /* Notes */ spelling fix

Seth Kasowitz — Mon, 29 Jan 2007 20:31:10 GMT

Notes: spelling fix

←Older revision		Revision as of 20:31, 29 January 2007
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	length product.		length product.

-	*Because you a priming your PCR off the plasmid, there will be a small fragment in every lane of your gel, even if a clone	+	*Because you are priming your PCR off the plasmid, there will be a small fragment in every lane of your gel, even if a clone

	had no insert. This also means that when looking on your gel you want to factor in that additional DNA when approximating		had no insert. This also means that when looking on your gel you want to factor in that additional DNA when approximating

Seth Kasowitz at 17:07, 29 December 2006

Seth Kasowitz — Fri, 29 Dec 2006 17:07:44 GMT

←Older revision		Revision as of 17:07, 29 December 2006
Line 27:		Line 27:

	==Procedure==		==Procedure==
-	#After performing your ligation reactions and transformations, plate the transformed cells on agar plates with an appropriate	+	#After performing your ligation reactions and transformations, plate the transformed cells on agar plates with an appropriate selectable marker. We typically use plasmids with ampicillin resistance.
-		+
-	selectable marker. We typically use plasmids with ampicillin resistance.	+
	#Grow plates overnight at 37°C		#Grow plates overnight at 37°C
	#The next day prepare a 96-well plate by adding 20 μL of MilliQ water to as many wells as you have colonies for.		#The next day prepare a 96-well plate by adding 20 μL of MilliQ water to as many wells as you have colonies for.
-	#Pick a colony and resuspend thoroughly in the water by pipetting	+	#Pick a colony and resuspend thoroughly in the water by pipetting. Cells will lyse over the next several minutes. Begin setting up your PCR immediately.
-	Cells will lyse over the next several minutes. Begin setting up your PCR immediately.	+
	#Prepare a master mix for PCR.		#Prepare a master mix for PCR.
	#Use 10μL of your lysed cells for template.		#Use 10μL of your lysed cells for template.
-	#As soon as you have taken your template DNA, add 50μL of LB + ampicillin (or whatever selection you are using) to the	+	#As soon as you have taken your template DNA, add 50μL of LB + ampicillin (or whatever selection you are using) to the remaining cells. Cover the plate with parafilm and place at 4°C.
-		+
-	remaining cells. Cover the plate with parafilm and place at 4°C.	+
	#Place samples in a thermal cycler and set program		#Place samples in a thermal cycler and set program
	#*When using M13, T7, or Sp6 primers we use an annealling temperature of 55°C.		#*When using M13, T7, or Sp6 primers we use an annealling temperature of 55°C.
-	#*Adjust extension time depending on your insert size. Be sure to keep in mind the amplified portion of your vector when	+	#*Adjust extension time depending on your insert size. Be sure to keep in mind the amplified portion of your vector when determining size.
-		+
-	determining size.	+
	#Pour a 1% agarose gel with ethidium bromide.		#Pour a 1% agarose gel with ethidium bromide.
	#Load PCR samples and an appropriate ladder (ie 2 log, 1kb, 100 bp)		#Load PCR samples and an appropriate ladder (ie 2 log, 1kb, 100 bp)

Seth Kasowitz: 12-29-2006: Page Created

Seth Kasowitz — Fri, 29 Dec 2006 16:18:00 GMT

12-29-2006: Page Created

New page

==Overview==
This allows you to screen a large number of inserts the day after completing a transformation, rather than waiting for overnight cultures and performing many unnecessary minipreps. A small amount of DNA is isolated directly from colonies on your plate. PCR amplification using primers
complementary to your plasmid lets you determine the presence and approximate size of inserts. The protocol works best for ligation reactions with known insert size(s).

==Materials==
*LB (or other suitable media)
*Selectable marker of choice
*Sterile deionized water

*[[PCR]] Reagents
**dNTPs
**Thermostable polymerase
**Buffer
***50 mM MgCl<sub>2</sub>
***500 mM KCl
***200 mM Tris-Hcl (ph 8.4)
**Primers
**Red Juice
***Cresol Red
***Sucrose
***MilliQ Water
***''<small>Note: Red Juice is a loading dye for agarose gels. Our lab adds it to PCRs as it saves a step between performing the reaction and running a sample on a gel. There is no negative impact to including this particular loading dye, and anecdotal evidence that it actually improves PCR results. Other loading dyes may have undesirable effects or completely impede the reaction.</small>''

*Agarose
*Ethidium Bromide
*DNA Marker

==Procedure==
#After performing your ligation reactions and transformations, plate the transformed cells on agar plates with an appropriate

selectable marker. We typically use plasmids with ampicillin resistance.
#Grow plates overnight at 37°C
#The next day prepare a 96-well plate by adding 20 μL of MilliQ water to as many wells as you have colonies for.
#Pick a colony and resuspend thoroughly in the water by pipetting
Cells will lyse over the next several minutes. Begin setting up your PCR immediately.
#Prepare a master mix for PCR.
#Use 10μL of your lysed cells for template.
#As soon as you have taken your template DNA, add 50μL of LB + ampicillin (or whatever selection you are using) to the

remaining cells. Cover the plate with parafilm and place at 4°C.
#Place samples in a thermal cycler and set program
#*When using M13, T7, or Sp6 primers we use an annealling temperature of 55°C.
#*Adjust extension time depending on your insert size. Be sure to keep in mind the amplified portion of your vector when

determining size.
#Pour a 1% agarose gel with ethidium bromide.
#Load PCR samples and an appropriate ladder (ie 2 log, 1kb, 100 bp)
#Electrophorese gel
#Visualize your gel and determine which clones have the desired insert.
#In a 14 ml culture tube, innoculate 2ml LB + ampicillin with 5μL of the cells from your 96-well plate.
#Grow overnight at 37°C and continue to [[miniprep]].

==Notes==
*This protocol is meant to be a time and resource saving method, but is not appropriate for every ligation and

transformation. Experiments that yield a large range of fragment sizes or very large fragments, such as BAC or genomic

digests (shotgun type cloning), may exceed the limitations of PCR or your particular ''taq''. Very large inserts may

necessitate such lengthy extension times that the PCR is no longer a time saving tool. Still, with a pool of fragments we

often find this technique useful as it lets us select one or two clones of each size, or choose the clone with our expected

length product.

*Because you a priming your PCR off the plasmid, there will be a small fragment in every lane of your gel, even if a clone

had no insert. This also means that when looking on your gel you want to factor in that additional DNA when approximating

fragment length. If a lane is completely empty there may have been a problem with your PCR. A template negative PCR reaction

is always a recommended control.

==Contact==
*[[User:Seth]]

[[Category:Protocol]]