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== Molly's Notes == | == Molly's Notes == | ||
| + | '''06/13/08'''<br> | ||
| + | |||
| + | 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. | ||
| + | |||
'''06/12/08'''<br> | '''06/12/08'''<br> | ||
Today was a busy day... <br> | Today was a busy day... <br> | ||
| - | 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, ligated with pBca1256, and transformed into competent cells. Finally, all were plated using sterile glass beads onto plates with Spec and placed in the 37 deg room to grow overnight. | + | 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. |
Revision as of 19:53, 13 June 2008
Molly's Notes
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.
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.
