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From 2013.igem.org

Amplification and purification of Del Genes from D. acidovorans DSM-39 genome

Goals

Our goal still remains to amplifiy the missing fragments from the Delftibactin cluster of D. Acidovorans. However, since we want to validate all fragments before we use them in a Gibson Assembly, and since last weeks restriction digests were either negative or inconclusive, these digests will be repeated. By the end of this week we will dicuss the further procedure and decide whether we again need to change our cloning strategy or not. Additionally a nested PCR of the sequence region surrounding DelOP might still be an option to provide specific template suitable for a PCR with our intended Gibson primers.

Results

PCRs from D.acidovorans DSM-39
Gene(s)	Fragment	Primer combination	Successful?
DelA DelB DelC DelD DelE DelF DelG	DelA-F	Primer FS_02 and FS_05
	DelA-G	Primer FS_02 and FS_26
	DelF-G	Primer FS_06 and FS_26
		Primer FS_20 and FS_26
		Primer FS_21 and FS_26
DelO DelP DelL	DelO-P	Primer FS_22 and FS_13
		Primer FS_22 and FS_13_short

Test restriction digests of PCR amplified fragments
Fragment	Primer	Digestion enzyme	Expected bands [bp]
DelA-E	Primer FS_02 and FS_03	BglII	2146, 1862, 1306
DelA-F	Primer FS_02 and FS_05	BglII	7229, 2146, 1862
DelF-G	Primer FS_21 and FS_26	XmaI	4268, 1202
DelG	Primer FS_08 and FS_23	ClaI	3291, 1936, 1303
DelO-P	Primer FS_22 and FS_13	EcoRI-HF	1883, 960