Team:Edinburgh/Lab Notebook

From 2011.igem.org

Revision as of 14:15, 9 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.

June 2: PCR seemed successful (though 2 bands were seen on the gel, 1 was the right size). We already have pSB1C3-RFP 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 Xgal- and 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 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.

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