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From 2013.igem.org

Amplification and purification of Del Genes from DSM-39

Goals

As our the amplifications of of the sequence region DelF-G did not progress last week, we decided to focus on this region in week 13. All combinations of initial and newly ordered primers will be tried with various PCR protocols. Furthermore, we tried to get higher amounts of the fragments for which protocols have already been established in order to have enough of each product to carry out suitable test restriction digests.

Results

PCRs from D.acidovorans DSM-39
Gene(s)	Fragment	Primer combination	Successful?
DelA DelB DelC DelD DelE DelF DelG	DelA-E	Primer FS_02 and FS_03
		Primer FS_02 and FS_24
	DelA-F	Primer FS_02 and FS_05
	DelA-G	Primer FS_02 and FS_26
		Primer FS_02 and FS_23
	DelE	Primer FS_04 and FS_24
	DelE-G	Primer FS_04 and FS_09
	DelF-G	Primer FS_06 and FS_07
		Primer FS_20 and FS_07
		Primer FS_20 and FS_23
		Primer FS_20 and FS_11_short
		Primer FS_21 and FS_07
		Primer FS_21 and FS_11_short
		Primer FS_21 and FS_23
		Primer FS_20 and FS_09
		Primer FS_21 and FS_09
	DelG	Primer FS_08 and FS_11_short
		Primer FS_08 and FS_23
DelO DelP	DelO-P	Primer FS_22 and FS_13
		Primer FS_22 and FS_13_short

Re-PCRs
Gene(s)	Fragment	Primer combination	Successful?
DelG	DelG	Primer FS_10 and FS_23
DelO DelP	DelO-P	Primer FS_22 and FS_13

Test restriction digests of PCR amplified fragments
Fragment	Primer	Digestion enzyme	Expected bands [bp]
DelA-E	Primer FS_02 and FS_03	EcoRI-HF	3054, 2260
DelA-F	Primer FS_02 and FS_05	EcoRI-HF	4624, 4354, 2260
DelE-G	Primer FS_04 and FS_07	PvuI-HF	6187, 4917
DelF-G	Primer FS_20 and FS_07	XmaI	4307, 879
	Primer FS_20 and FS_07	ClaI	2420, 1519, 1247
DelG	Primer FS_08 and FS_23	BglII	3291, 1936, 1303
DelO-P	Primer FS_22 and FS_13_short	EcoRI-HF	1883, 960