Shipping Vector Cloning
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.
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.
Figure 1: A 1.5% agarose gel showing the results of a PCR from gBlock of: Lanes 3 and 4 - part V300 with length 1988 Lanes 6 and 7 - part V400 of length 1826, two parts that were not submitted to the registry due to illegal restriction sites
We then digested the parts and ligated them into the shipping vector, which we obtained from a stock from last year’s team. We chose this over linearised backbone from the distribution kit because it was already at a high concentration and contained a part at around 1.4kb, which was easy to separate on an agarose gel in order to only get the cut vector. We had difficulties with vector self-ligation, so we began to treat the digestions with calf intestinal phosphatase. This almost entirely eliminated the issue, and we had fewer colonies on our plate but a much higher ratio of correct colonies.
Figure 2: Examples of some plates from transformations, with colonies ringed for inoculations
This was then transformed and grown-up in order to obtain a high-yield miniprep stock of plasmid. The minipreps were tested by digesting them and running them on an agarose gel. We experimented with colony PCR, and have a protocol, in order to select for the correct colonies before inoculating, however we found test-digesting to be more reliable.
Once the test digest showed the part had inserted the sequences were confirmed by sequencing.
Figure 3: An example sequencing trace
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!
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.
Numbering SystemWe also implemented a numbering system for our parts, so that eppendorfs, plates and test-tubes were all labelled correctly. It also took up less room than writing the full part name out every time! We taped this up in the lab so that everyone could see what to call the bit they were working on.