Cloning Checklist - Revision history

Christopher C Vanlang at 10:18, 22 December 2011

2011-12-22T10:18:46Z

←Older revision		Revision as of 10:18, 22 December 2011
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	*'''DO''' make sure your primers have similar <math>T_m</math>s.		*'''DO''' make sure your primers have similar <math>T_m</math>s.
	**They should be within 5°C of each other. Set your PCR annealing temperature to 5°C less than your lowest <math>T_m</math>. Only calculate the <math>T_m</math> for the sections which anneal.		**They should be within 5°C of each other. Set your PCR annealing temperature to 5°C less than your lowest <math>T_m</math>. Only calculate the <math>T_m</math> for the sections which anneal.
		+
		+	[[Category:Protocol]]
		+	[[Category:Cloning]]
		+	[[Category:DNA]]
		+	[[Category:In vitro]]

Robwarden: /* Choosing Restriction Enzymes */

2009-03-25T15:54:35Z

Choosing Restriction Enzymes

←Older revision		Revision as of 15:54, 25 March 2009
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	**Technically they work. Practically, not so well... Cloning will go much smoother with sticky ends, so avoid blunt ends if possible. Even doing a mutation to add a sticky restriction site will probably save you time.		**Technically they work. Practically, not so well... Cloning will go much smoother with sticky ends, so avoid blunt ends if possible. Even doing a mutation to add a sticky restriction site will probably save you time.
	*'''DO''' use enzymes with different sticky ends.		*'''DO''' use enzymes with different sticky ends.
-	**This will allow you to make sure your DNA ends up in the vector in the right direction. It will also allow the backbone to close on itself (can be fixed by dephosphorlyating the 5' end of the backbone).	+	**This will allow you to make sure your DNA ends up in the vector in the right direction. It will also not allow the backbone to close on itself (can be fixed by dephosphorlyating the 5' end of the backbone).
	*'''DO''' check for methylation sites.		*'''DO''' check for methylation sites.
	*''dam'' methyltransferase: GATC		*''dam'' methyltransferase: GATC

Robwarden: /* Overall Plan */

2008-06-17T18:42:54Z

Overall Plan

←Older revision		Revision as of 18:42, 17 June 2008
Line 11:		Line 11:
	**Mutagenesis is simple and reliable and can save LOTS of time. Use it to switch up restriction sites throughout the vector. But don't forget, silent mutations aren't always silent, they effect translation and folding kinetics, so try to make silent mutations that have similar codon bias. [http://openwetware.org/wiki/Escherichia_coli/Codon_usage OpenWetWare] has a good codon bias table.		**Mutagenesis is simple and reliable and can save LOTS of time. Use it to switch up restriction sites throughout the vector. But don't forget, silent mutations aren't always silent, they effect translation and folding kinetics, so try to make silent mutations that have similar codon bias. [http://openwetware.org/wiki/Escherichia_coli/Codon_usage OpenWetWare] has a good codon bias table.
	*'''DO NOT''' interfere with upstream regulatory regions.		*'''DO NOT''' interfere with upstream regulatory regions.
-	**Unless you mean to ~~changing~~ them, too. Promoters have important regions about 35bp and 10bp before the start codon. And don't forget about the Shine-Dalgarno sequence (consensus in ''E. Coli'': AGGAGGA) which is needed for mRNA to find the ribosome.	+	**Unless you mean to change them, too. Promoters have important regions about 35bp and 10bp before the start codon. And don't forget about the Shine-Dalgarno sequence (consensus in ''E. Coli'': AGGAGGA) which is needed for mRNA to find the ribosome.
	*'''DO''' gel purify after restriction reactions.		*'''DO''' gel purify after restriction reactions.
	**You'll lose more DNA than with a PCR cleanup kit, but you're more likely to get what you want if you get rid of the DNA pieces you don't want.		**You'll lose more DNA than with a PCR cleanup kit, but you're more likely to get what you want if you get rid of the DNA pieces you don't want.

Robwarden: /* Overall Plan */

2008-06-13T17:05:34Z

Overall Plan

←Older revision		Revision as of 17:05, 13 June 2008
Line 14:		Line 14:
	*'''DO''' gel purify after restriction reactions.		*'''DO''' gel purify after restriction reactions.
	**You'll lose more DNA than with a PCR cleanup kit, but you're more likely to get what you want if you get rid of the DNA pieces you don't want.		**You'll lose more DNA than with a PCR cleanup kit, but you're more likely to get what you want if you get rid of the DNA pieces you don't want.
		+	*'''DO NOT''' change the reading frame of the protein.
		+	**Make sure to translate your final product before ordering any primers. This will make sure you kept the right reading frame throughout your construct.

	==Choosing Restriction Enzymes==		==Choosing Restriction Enzymes==

Robwarden: /* Overall Plan */

2008-06-04T17:45:00Z

Overall Plan

←Older revision		Revision as of 17:45, 4 June 2008
Line 12:		Line 12:
	*'''DO NOT''' interfere with upstream regulatory regions.		*'''DO NOT''' interfere with upstream regulatory regions.
	**Unless you mean to changing them, too. Promoters have important regions about 35bp and 10bp before the start codon. And don't forget about the Shine-Dalgarno sequence (consensus in ''E. Coli'': AGGAGGA) which is needed for mRNA to find the ribosome.		**Unless you mean to changing them, too. Promoters have important regions about 35bp and 10bp before the start codon. And don't forget about the Shine-Dalgarno sequence (consensus in ''E. Coli'': AGGAGGA) which is needed for mRNA to find the ribosome.
		+	*'''DO''' gel purify after restriction reactions.
		+	**You'll lose more DNA than with a PCR cleanup kit, but you're more likely to get what you want if you get rid of the DNA pieces you don't want.

	==Choosing Restriction Enzymes==		==Choosing Restriction Enzymes==

Robwarden: /* PCR */

2008-06-04T17:36:54Z

PCR

←Older revision		Revision as of 17:36, 4 June 2008
Line 33:		Line 33:
	**This will make the primer 'sticky' and lots of nonspecific binding/polymerization will occur. 50% is a good G/C target. This is especially important at the 3' end of the molecule.		**This will make the primer 'sticky' and lots of nonspecific binding/polymerization will occur. 50% is a good G/C target. This is especially important at the 3' end of the molecule.
	*'''DO''' make sure your primers have similar <math>T_m</math>s.		*'''DO''' make sure your primers have similar <math>T_m</math>s.
-	**They should be within 5°C of each other. Set your PCR annealing temperature to 5°C less than your lowest <math>T_m</math>.	+	**They should be within 5°C of each other. Set your PCR annealing temperature to 5°C less than your lowest <math>T_m</math>. Only calculate the <math>T_m</math> for the sections which anneal.

Robwarden: /* Choosing Restriction Enzymes */

2008-06-04T17:32:06Z

Choosing Restriction Enzymes

←Older revision		Revision as of 17:32, 4 June 2008
Line 19:		Line 19:
	**Technically they work. Practically, not so well... Cloning will go much smoother with sticky ends, so avoid blunt ends if possible. Even doing a mutation to add a sticky restriction site will probably save you time.		**Technically they work. Practically, not so well... Cloning will go much smoother with sticky ends, so avoid blunt ends if possible. Even doing a mutation to add a sticky restriction site will probably save you time.
	*'''DO''' use enzymes with different sticky ends.		*'''DO''' use enzymes with different sticky ends.
-	**This will allow you to make sure your DNA ends up in the vector in the right direction.	+	**This will allow you to make sure your DNA ends up in the vector in the right direction. It will also allow the backbone to close on itself (can be fixed by dephosphorlyating the 5' end of the backbone).
	*'''DO''' check for methylation sites.		*'''DO''' check for methylation sites.
	*''dam'' methyltransferase: GATC		*''dam'' methyltransferase: GATC

Robwarden: /* PCR */

2008-06-04T17:21:35Z

PCR

←Older revision		Revision as of 17:21, 4 June 2008
Line 30:		Line 30:
	*'''DO''' make sure your primer's 3' nucleotide is G or C.		*'''DO''' make sure your primer's 3' nucleotide is G or C.
	**G and C bind tighter than A and T. Ending with G/C gives the polymerase a good base to start polymerization.		**G and C bind tighter than A and T. Ending with G/C gives the polymerase a good base to start polymerization.
-	*'''DO NOT''' make the ~~3' end~~ too G/C rich.	+	*'''DO NOT''' make the primer too G/C rich.
-	**This will make the primer 'sticky' and lots of nonspecific binding/polymerization will occur. 50% is a good G/C target.	+	**This will make the primer 'sticky' and lots of nonspecific binding/polymerization will occur. 50% is a good G/C target. This is especially important at the 3' end of the molecule.
		+	*'''DO''' make sure your primers have similar <math>T_m</math>s.
		+	**They should be within 5°C of each other. Set your PCR annealing temperature to 5°C less than your lowest <math>T_m</math>.

Robwarden: New page: =''E. Coli'' Cloning Checklist= This is a rough guide to the DOs and DO NOTs of cloning in ''E. Coli''. It was assembled based on problems I've encountered while attempting restriction d...

2008-06-04T17:11:24Z

New page: =''E. Coli'' Cloning Checklist= This is a rough guide to the DOs and DO NOTs of cloning in ''E. Coli''. It was assembled based on problems I've encountered while attempting restriction d...

New page

=''E. Coli'' Cloning Checklist=

This is a rough guide to the DOs and DO NOTs of cloning in ''E. Coli''. It was assembled based on problems I've encountered while attempting restriction digests, ligations, and PCR.

==Overall Plan==
*'''DO''' create/destroy restriction sites.
**This will allow for better verification of what you've done. It's just easier to tell the difference between the number of bands than the size of bands.
*'''DO''' plan validation digests from the beginning.
**If you plan these while you're choosing enzymes, you can avoid having to try to tell the difference between 1300bp and 1350bp. You can have less difference between low molecular weight fragments (~100 bp if fragments are < 1kb). However, high molecular weight fragments require large differences (sometimes more than 2 or 3 kb if <3 kb). If you can clearly tell the difference between two lenghts on your ladder, it will probably work for a digest, too.
*'''DO''' take advantage of mutagenesis.
**Mutagenesis is simple and reliable and can save LOTS of time. Use it to switch up restriction sites throughout the vector. But don't forget, silent mutations aren't always silent, they effect translation and folding kinetics, so try to make silent mutations that have similar codon bias. [http://openwetware.org/wiki/Escherichia_coli/Codon_usage OpenWetWare] has a good codon bias table.
*'''DO NOT''' interfere with upstream regulatory regions.
**Unless you mean to changing them, too. Promoters have important regions about 35bp and 10bp before the start codon. And don't forget about the Shine-Dalgarno sequence (consensus in ''E. Coli'': AGGAGGA) which is needed for mRNA to find the ribosome.

==Choosing Restriction Enzymes==
*'''DO''' check for enzyme uniqueness in both the backbone and insert DNA.
**It's simple, but a terrible thing if you forget to... Restriction sites can be created/destroyed before cloning by mutagenesis.
*'''DO NOT''' create blunt ends.
**Technically they work. Practically, not so well... Cloning will go much smoother with sticky ends, so avoid blunt ends if possible. Even doing a mutation to add a sticky restriction site will probably save you time.
*'''DO''' use enzymes with different sticky ends.
**This will allow you to make sure your DNA ends up in the vector in the right direction.
*'''DO''' check for methylation sites.
**''dam'' methyltransferase: GA*TC
**''dcm'' methyltransferase: CC*AGG, CC*TGG
**Methylation inhibits restriction enzymes. [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/dam_dcm_methylases_of_ecoli.asp NEB] lists ''dam'' or ''dcm'' sensitivity for each of their enzymes. If your enzyme is on this list, make sure there is no overlapping methylation site. If there is, fix it by mutagenesis or do your cloning in ''dam-/dcm- E. Coli'' strains (sold by [http://www.neb.com/nebecomm/products/productC2925.asp NEB]).

==PCR==
*'''DO''' include 8-10 random nucleotides between the end of the PCR fragment and a restriction site.
**Restriction enzymes need DNA to 'sit' on so that they can cut. If the restriction site is too close to the end, the enzyme will fall off the DNA before it's enzymatic activity can occur. My favorite filler is CATGTAGC.
*'''DO''' make sure your primer's 3' nucleotide is G or C.
**G and C bind tighter than A and T. Ending with G/C gives the polymerase a good base to start polymerization.
*'''DO NOT''' make the 3' end too G/C rich.
**This will make the primer 'sticky' and lots of nonspecific binding/polymerization will occur. 50% is a good G/C target.