Team:Heidelberg/Templates/Del week14 overview

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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.


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
Heidelberg Tilde.png
DelA-G Primer FS_02 and FS_26
Heidelberg Red x.svg.png
DelF-G Primer FS_06 and FS_26
Heidelberg Red x.svg.png
Primer FS_20 and FS_26
Heidelberg Red x.svg.png
Primer FS_21 and FS_26
Heidelberg Tilde.png
DelO
DelP

DelL
DelO-P Primer FS_22 and FS_13
Heidelberg Tilde.png
Primer FS_22 and FS_13_short
Heidelberg Tilde.png



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