Team:Edinburgh/Lab Notebook

From 2011.igem.org

Revision as of 17:49, 10 June 2011

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Note: Protocols were generally similar to those found on Open Wetware's [http://www.openwetware.org/wiki/French_Lab pages] dedicated to Chris French's Lab.

June 1: Began work to add the Plac-lacZα sequence to the pSB1C3 vector. PCR was used to introduce the BglII restriction site (so the final thing becomes E-X-B-Plac-lacZα-S-P). This gives us a vector that we like. For the principles behind the use of lacZα see [http://utminers.utep.edu/rwebb/html/alpha_complementation.html here]. It will enable us to determine whether inserts are present using blue/white screening. In the future, vectors in which the desired part successfully replaces lacZα will be white.

If I [AC] understand correctly, having a BglII site in the vector is useful as it means any DNA sequence can easily have the sites needed for incorporation into the vector added (by PCR) at both ends, whereas in the standard RFC10 system the two most obvious sites (XbaI and SpeI, which come immediately before and after the BioBrick itself) cannot actually be used this way, because they generate compatible sticky ends, meaning the product would circularise.

June 2: PCR seemed successful (though 2 bands were seen on the gel, 1 was the right size). We already have pSB1C3-RFP (Red Fluorescent Protein) so we cut out the RFP (using restriction enzymes XbaI and PstI) and inserted the new sequence.

June 3: After transformation of competent cells we plated onto an [http://en.wikipedia.org/wiki/X-gal Xgal]- and [http://en.wikipedia.org/wiki/IPTG IPTG]- containing media.

At this point we also prepared about 15 plates of agar, with chloramphenicol (which the pSB1C3 vector provides resistance to) as well as Xgal and IPTG for later blue/white screening.

June 6: Colonies that have successfully lost RFP and gained lacZα should be blue (on this media). We found several such colonies (though many others were red and some were white). Six blue colonies were selected, replated and grown for 6 hours on the media mentioned above. Some cells were then transferred to bottles of media for further growth.

June 7: Plasmid DNA from the bottled cultures was purified (by the "miniprep" protocol). We digested it with EcoRI to linearise it, and then ran it on a gel. All six cultures showed a band of the correct size (about 2600 b.p.) but we will send some DNA for sequencing.

June 8: Clones 1 and 2 of pSB1C3-lacZα were selected for sequencing. Four tubes were sent to the university's sequencing service:

1. Clone 1, Forward
2. Clone 1, Reverse
3. Clone 2, Forward
4. Clone 2, Reverse

We expect results on the 10th (Friday).

We also began PCR to add the BglII restriction site to some RFP and YFP (Yellow Fluorescent Protein) coding sequences (with no promoters). Bands on a gel showed that PCR apparently worked for the YFP but not the RFP. Nevertheless, we purified both and placed them in the pink box in the fridge.

June 9: We started to insert YFP into our new vector. We digested both pSB1C3-lacZα and our YFP product with BglII and SpeI. We then started a ligation reaction and left it overnight. There was some concern about contamination as the reaction mixture picked up a yellowish colour, possibly from dirty (unused but old) pipette tips.

If successful, some plasmids should become: E-X-B-YFP-S-P

Since there's no promoter and no lacZ gene, these should give white colonies on Xgal+IPTG media. We expect to do the transformations and plating tomorrow.

June 10: We transformed and plated our pSB1C3 + YFP (+ PlaclacZα, which we are trying to excise) onto Xgal+IPTG plates. There are 4 plates; we decided to investigate whether the heat shock part of the transformation protocol has any effect, thus 2 were not heat shocked.

We also got back sequences of our vector. We will have to figure out what software to use and how, but Eugene says the sequence from clone 2 appears better than that of clone 1. Apparently [http://www.geospiza.com/Products/finchtv.shtml FinchTV] is freely available software we can use. Allan has added his best guess for the exact sequence of clone 2 to the sequences page.

We should probably use our pSB1C3-Plac-lacZα clone 2 as our vector of choice in the future.

This was the old Navbox for Edinburgh; now it's obsolete...