Shipping Vector Cloning
Introduction
We have included this page to summarise the techniques and results we had in cloning our parts into the pSB1C3. For a detailed look at the protocols themselves and every step of the process please visit our protocols page.
Cloning Steps
For each part we began by cloning it into pSB1C3, the iGEM shipping vector. The protocols we used for this are on our protocols page. We began by amplifying our parts out of the gBlocks that IDT had sent us by PCR. We experimented with various conditions before achieving the conditions specified in the PCR protocol. We found we needed to dilute the DNA 1:10 and add 1ul of DMSO to the reaction mixture in order to achieve the best results.![](https://static.igem.org/mediawiki/2017/3/3c/T--Oxford--Results--Cloningpcrgel.png)
![](https://static.igem.org/mediawiki/2017/8/86/T--Oxford--Results--Testdigestgel.png)
![](https://static.igem.org/mediawiki/2017/b/b3/T--Oxford--Results--sequencingtrace.png)
Tips for Successful Cloning
PCR Masterlist Spreadsheet
By the end of our project we had done a variety of different PCRs, with different primers, DNA templates, and conditions, therefore we made a spreadsheet in order to keep track of them all, so members of the team could generate their own spreadsheet and print it out if they were doing a large number of reactions at once. We recommend that other teams do this early on, as we could have saved a lot of time by implementing this system from the start of the project!![](https://static.igem.org/mediawiki/2017/0/0e/T--Oxford--Results--pcrspreadsheet.png)
Double Digest Masterlist
We also made our own version of the NEB double-digest poster, with which buffers we could use with which restriction enzymes, because although HF-versions of enzymes and Cutsmart has been around for a while, there are still lots of non-HF versions of the enzymes around in our lab. Our version was simplified and only included the enzymes we were using frequently, this saved a lot of time when running lots of digestions simultaneously, this is something else we recommend teams do early on.![](https://static.igem.org/mediawiki/2017/4/4d/T--Oxford--Results--digestmasterlist.png)