Team:Heidelberg/Templates/Del week14 overview
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==Amplification and purification of Del Genes from ''D. acidovorans'' DSM-39 genome== | ==Amplification and purification of Del Genes from ''D. acidovorans'' DSM-39 genome== | ||
- | ===Goals=== | + | ====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. | 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. | 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. | ||
<br/> | <br/> | ||
- | + | ====Results==== | |
+ | The following table summarizes the outcomes of this weeks PCRs | ||
{| class="wikitable" | {| class="wikitable" |
Revision as of 18:15, 4 October 2013
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
The following table summarizes the outcomes of this weeks PCRs
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 |