From 2007.igem.org

Digestion reactions

Redigestion reactions were performed on several PCR products already digested. This was done because of owr persistant inability to clone any PCR product.

2x reaction mixes were prepared:

• XbaI+PstI digestion reactions (buffer NEB3)
• EcoRI+PstI (NEBU buffer)
• SpeI+PstI (NEB2)

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description	vol. left for dig.
D19'	BB dig. lox66DapAsubtilis	lox66-DapAsubtilis P6' PCR product	EcoRI	PstI	~960	PCR product digested for cloning as a BioBrick	20
D21'	bw-insert lox66-DapAsubtilis	lox66-DapAsubtilis P6' PCR product	XbaI	PstI	~960	lox66-DapAsubtilis P6' PCR product	25
D27'	lox71-ftsZ bw-insert	lox71-ftsZ PCR product P7	XbaI	PstI		lox71-ftsZ(unmutated) ready for use as a backward insert	30
D28	RBS-DapAColi BB	P8 (RBS-DapAColi)	EcoRI	PstI	~950	Biobrick	15
D29	RBS-DapAColi	P8 (RBS-DapAColi)	XbaI	PstI	~950	Backward insert	13
D30	RBS-DapASubtilis BB	P9 (RBS-DapASubtilis)	EcoRI	PstI	~950	Biobrick	14
D31	RBS-DapASubtilis	P9 (RBS-DapASubtilis)	XbaI	PstI	~950	Backward insert	14
D32	Lox71-FtsA-FtsZ	P10 Lox71-FtsA-FtsZ	XbaI	PstI	~2600	Backward insert - EcoRI site still presents : we can't use EcoRI	18
D34	lox66	L9' lox66	SpeI	PstI		Biobrick plasmid carrying lox66 digested as a BV	26
D35	lox71	L10' lox71	SpeI	PstI		Biobrick plasmid carrying lox71 digested as a BV	33
D36	lox71-KmR-lox66	P11 lox71-KmR-lox66	EcoRI	PstI		lox71-KmR-lox66 digested as a biobrick insert	28

Also, new digestion reactions were performed:

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	Size	Description
D18	BB dig. lox66DapAE.coli	lox66-DapAE.coli P5 PCR product	EcoRI	PstI	~960	PCR product digested for cloning as a BioBrick
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert
D20	bw-insert lox66-DapAE.coli	lox66-DapAE.coli P5 PCR product	XbaI	PstI	~960	PCR product digested for usage as a backward insert

PCR on colonies

• P6 : again (1,7) + new (9,10,11,12)
• P7 : again (1) + new (9,10,11,12,13,14,15)
• P8 : again (1,3,5,7,8) + new (9,10,11)
• P10 : again (old ones 1,5,6) + new (9,10,11,12,13)