In the lab: We discussed a lot about what to do with making more vector because we’d really been struggling to get any descent amplifications of it from the PCR. So we looked at everything we had and decided to start from the very beginning with the circular plasmid rather than trying to make more of the linear vector directly from some other linear vector reserves (because we weren’t sure exactly what our linear vector was ie was it suitable for PCR or not).

We set up a PCR of the circular plasmid then when that had finished and we’d checked it’d worked by gel electrophoresis we went on to do transformation. We put the circular plasmid into e.coli cells and grew them up on selective media over night. We also did homologous recombination and plated up one of our genes which we new was correct and would work (gene X) to use as a positive control.


In the lab: Adam came up with a new idea for trying to get gene V amplyfied by PCR. We made serial dilutions of the forward and reverse primes and used lots of different combinations of them in the PCR mixes to see if we could get a stronger band of our desired gene and less of the little dimers which we kept getting loads of and which were getting in the way.

We did the PCR and ran it on a gel to see the results. We had a major breakthrough; the results were really interesting and clearly showed that it was the forward primer which was mostly resposible for the dimers. In the mixes where there was less forward dimer to reverse primer we had really strong bands of our gene and a lot less dimers. This was very exciting as it was the best result we’d had so far for gene V! The plates of our plasmids, which we’d left to grow overnight had loads of colonies on them which was great so we picked a few of these and grew them in liquid media overnight for digetion the next day.


In the lab: We pelleted and restriction digested our plasmid which had grown in liquid media overnight to see if it was infact the right plasmid. The gel electrophoresis showed that it was the correct plasmid so we went on to do a different digestion of the plasmid to make it linear so that we could use it for the homologous recombination (this incubates overnight).  We then decided to try and get a strong, clean source of our gene V. So we ran out some of the better PCR tests from the day before on a gel to do a gel isolation of all of them.

We added all of these gel isolations together so that we had a superconcentrated gel isolate of our gene. After we’d done this we ran this out on a gel to check that we’d got a nice clean source of our gene V and that we had enough of it ie. Hadn’t lost too much during the gel isolation proccess. The result looked very good so we now have a strong, clean source of gene V ready to go on to homolgous recombination! 


In the lab: The vector should have been cut overnight making it into an linear vector for us to use for homologous recombination. We ran the vector that had been digested overnight on a gel to see if it had been cut (using an uncut vector as a control). The gel electrophoresis showed that the vector had been cut so then we did a PCR of the vector so that it had the correct end (dictated by our primers) and now we have a linear vector to use in our homologous recombination.


In the lab: We cleaned up the linear vector we had made and measured its concentration using the nanodrop spectrophotometer but we couldn’t set up the homologous recombination yet because it was the weekend the next day. Megan in my lab was planning to come in on Sunday anyway so she will said she’d set it up on Sunday so its ready for Monday instead. So an early finish today!

After the lab: Fiona came back to mine for the weekend as Katie and Liz are both away so we thought it’d be better if she stayed at mine for a bit becaseu you can walk to more places from mine. We decided to get Ben & Jerry’s on the way home. We both got a hot fudge sundae. It was really lovely, but so chocolatly I felt completely chocolated out afterwards! We then had pizza for dinner whilst we watched the London 2012 Olympic opening ceremony.


We went to visit the North Carolina Museum of Art for the day. We walked there along footpaths and it was absolutly boiling hot but once we got into the Museum Park which surrounded the Museum there was lots of other little paths that went into a wood under some trees so that was a nice relief.

We saw half of the outside art pieces, some of which were really interesting there was a particularly intersting one called the Cloud Chamber. We got to the main museum buildings and there was loads of stuff in there and we spent ages walking around the exhibits. There was some more outside things such as a really pretty pond/ water feature with lilies and damselflies flying around it.

After we’d seen everything we walked home via a slightly different route to see the other half of the outside art. Then finally we got home; we were so hot we went inside to cool off before doing anything else. When we’d cooled down we went to The Waffle House for dinner.


We had a lovely lie in before making our way to Bojangles Famous Chicken ‘n Biscuits. We both got The Cajun fillet Biscuit combo with fries. On the way back from Bojangles we stopped off at a supermarket to buy some goodies!!


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