Template:Team:UC Berkeley/Notebook/Molly notes

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Molly's Notes

06/20/08

I retrieved my gel-isolated PCR products of K112200, K112201, K112204, and K112205 (using the real mea002 oligo!) and digested them, did a zymo clean-up, ligated them with twice the recommended amount of pBca1256 re-digest since it was dilute, transformed and plated them. They are incubating in Chris's lab.

I also got sequencing data on K112207-12,229. K112229 turned out to indeed be the sequencing for K112230, and it was perfect aside from a strange deletion of A in the EcoRI site, but it looks like it's just a bad read error. K112207-12 were supposed to be run with G00101, but were instead run again with ca998... but they do have enough plasmid to do another reverse read, so that's good. I'll be expecting that data soon.

Today, I also did a mini-prep on K112219, K112220, K112223, and K112224 colonies I picked/grew up in a culture tube yesterday. The samples will be sequenced under the names MEA219-224.

06/19/08

I checked plates K112205, K112219, K112220, K112223, and K112224 today and threw out K112205 since it used the faulty mea002 oligo. LOTS (around 50% in some cases) of the colonies are red (parent vector), indicating Aron's vector digest probably wasn't all that pure. The K112212 plate has a ton of very very tiny colonies growing, so we parafilmed it an put it in the fridge. The glass culture tube was dumped into the 24-well block with Bing and Aron's samples and Bing mini-prepped them. At the end of the day, I picked colonies from K112219, K112220, K112223, and K112224 and grew them in 5 mL of 2XYT/Spec in glass culture tubes and left them overnight in Chris's shaker.

Because there were many red colonies, we decided to re-digest the vector digest that Aron made with the newer digest that Bing and Aron made. We let them digest for 2 hours instead of 1. Then I ran all of the sample on a clone-well E-gel to isolate the digested vector. The bands were surprisingly light and comet-like, suggestive of over-loading. However, Chris looked at it and said it looked fine, so I went ahead and isolated that and put it in the freezer.

Also, today Chris gave us a tutorial on how to use Adobe Illustrator and someone took a group pic.

My new oligo mea002 came today! I diluted it to 100 uM and made a 10 uM stock with 5 ul of oligo and 45 ul of water. I used this to set up a PCR of K112200, K112201, K112204, and K112205 and ran them with two of Aron's samples with program 55. Aron stayed late today and said he would gel-isolate my product as well as his own and store mine in the freezer. (Thanks!)

06/18/08

So I took out my PCR products, ran/isolated them on an E-gel. I took my K112205 sample out of the freezer and included them with my isolated K112219, K112220, K112223, and K112224 samples in digestion, zymo clean-up, ligation, transformation, and plating. Chris had run out of his digested pBca1256 vectors, so we used the ones Aron digested the other day. We included one of Bing's samples that had previously worked as a control. These were left to incubate at 37 C in Chris's lab.

Terry came in today and talked about the modeling of holin and antiholin. I still need to start looking for papers and reading over the ones that Terry already found.

Sequencing data came in today as well. All the integrase reverse-sequencing reads (done after the sequencing lab re-amplified the product we already had) gave bad data 200 bp after the oligo, but were otherwise perfect. We mini-prepped the stock clones (K112207-K112211) but there wasn't any for K112212. So we took the plasmids from the clone we sent in originally and re-transformed them back into bacteria. Part of the transformation was plated, and part was grown up in a glass culture tube containing spec. Both are in Chris's lab. We also picked a new (third) colony for K112229 since the sequencing data was bad again for this. K112229 is in A1 of a 24-well block along with Bing and Aron's stuff. K112207 is in B3, K112208 is in B4, K112210 is in B5, K112209 is in B6, and K112211 is in C1.

The sequencing data for K112200, K112201, K112204, K112216, and K112229 also arrived today (MA001, MA002, MA003, MA005, and MA004 respectively). The read for colony 2 of K112204 was a bad read, as was the read for K112229. K112216 proved perfect. K112200 and K112201 showed a common point mutation changing Asn to Lys, which was also identical to the mutation in the first clone of K112204. So I decided to check out their common oligo, and it turns out that Jin had erroneously added an extra C to the 3' end of this reverse oligo which cased the fatal point mutation from a T to a G. <shakes fist indignantly> This means I will have to start over on K112200, K112201, K112204, and K112205.

06/17/08

My new (correct) oligo mea014 arrived today, so I set up PCR for K112219, K112220, K112223, and K112224. Most of the day otherwise was spent discussing composite parts and such. Bing and Aron picked new colonies for the ones that didn't work (for me that was K112200, K112201, K112204, K112216, and K112229).

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.

Molly's Notebook

@@ Line 3: / Line 3: @@
 '''06/20/08'''<br>
-I retrieved my gel-isolated PCR products of K112200, K112201, K112204, and K112205 (using the real mea002 oligo!) and digested them.
+I retrieved my gel-isolated PCR products of K112200, K112201, K112204, and K112205 (using the real mea002 oligo!) and digested them, did a zymo clean-up, ligated them with twice the recommended amount of pBca1256 re-digest since it was dilute, transformed and plated them. They are incubating in Chris's lab.
+I also got sequencing data on K112207-12,229. K112229 turned out to indeed be the sequencing for K112230, and it was perfect aside from a strange deletion of A in the EcoRI site, but it looks like it's just a bad read error. K112207-12 were supposed to be run with G00101, but were instead run again with ca998... but they do have enough plasmid to do another reverse read, so that's good. I'll be expecting that data soon.
+Today, I also did a mini-prep on K112219, K112220, K112223, and K112224 colonies I picked/grew up in a culture tube yesterday. The samples will be sequenced under the names MEA219-224.