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== Molly's Notes == | == Molly's Notes == | ||
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| + | '''06/16/08''' <br> | ||
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| + | Today I analyzed all of the sequencing data that Bing and Jin sent in over the weekend. See my sequencing log page for the results. Some of the sequencing files seemed to be switched around, so Jin is having them re-sequence some of them. The ones that were "perfect" were transformed into competent cells. All of the integrase basic parts were also sent back to be sequenced with the reverse oligo G00101 since they're over 1000 bp. The integrase template also seemed to have a silent mutation, since the exact same one showed up in all 6 basic parts. | ||
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| + | Bing and Aron also picked other colonies for the sequencing data that didn't work. | ||
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'''06/13/08'''<br> | '''06/13/08'''<br> | ||
Revision as of 17:13, 17 June 2008
Molly's Notes
06/16/08
Today I analyzed all of the sequencing data that Bing and Jin sent in over the weekend. See my sequencing log page for the results. Some of the sequencing files seemed to be switched around, so Jin is having them re-sequence some of them. The ones that were "perfect" were transformed into competent cells. All of the integrase basic parts were also sent back to be sequenced with the reverse oligo G00101 since they're over 1000 bp. The integrase template also seemed to have a silent mutation, since the exact same one showed up in all 6 basic parts.
Bing and Aron also picked other colonies for the sequencing data that didn't work.
06/13/08
So I ran K112205, K112219, K112220, K112223, and K112224 with and without DMSO on E gels. K112205 with and without DMSA both worked, and are in the freezer in tubes labeled A6 with and without a black filled-in dot for with and without DMSO. However, none of the others did work. We checked it out and discovered that mea014 was not copied and pasted correctly in the oligos to order spreadsheet, so it was actually just another forward oligo rather than the reverse that was crucial to all four parts that didn't work. Jin will order it on Monday in case anyone else needs more between now and then. I've decided to wait on my successful PCR with K112205 until I have the other four done.
All of my plates looked good (even the contaminated one, K1122209!) so we made 2YT media, picked colonies, and put them in a 96 well plate-thing with 1 mL of media each. We put it on the shaker table in the 37 C room to grow overnight. Tomorrow, Bing will come in and do a miniprep/send them off to be sequenced.
06/12/08
Today was a busy day...
First, I ran all 24 of my PCRed parts (K112200-K112224) again and got acceptable products for all but K112205, K112219, K112220, K112223, and K112224. So, I ran these five again with and without DMSO with program 45 and will run an E gel tomorrow. The rest of my samples were digested with EcoRI and BamHI, cleaned up with the zymo kit, ligated with pBca1256, and transformed into competent cells. It should be noted that K112209 was submerged in the ice-water bath before heat-shock, and was thus probably contaminated. My wobble parts (K112225-K112230) were similarly prepared and plated. Finally, all were plated using sterile glass beads onto plates with Spec and placed in the 37 deg room to grow overnight.
06/11/08
All of my oligos came today (yay!) I successfully made all my wobble prepro parts (i.e. K112225-K112230) today, purified them on an cloning E-gel (next time remember to keep filling the second wells to get the max amount of product!) I got about 15 uL of each product. Next, these were digested with EcoR1 and BamH1, and incubated at 37 deg C for 1 hour. Finally, the DNA was purified using the Zymo kit.
The master mix used for all 24 of my other parts (K112200-K112224) had too much of the dNTP mix, and running it on an analytical E-gel showed that the was no PCR product. At the end of the day, we tried again and put them in the thermocyclers to be purified tomorrow.
