Team:Johns Hopkins/Notebook/GROUP 2: MATa Specific-promoters

GROUP 2: MATa Specific-promoters
Date: September 7, 2008 Status report by: Allison and Nate Part no.: BBa_K110008 -> BBa_K110016 Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively. Since the restriction digestions have been unclear as to whether or not the ligation of MFA1 and STE2 into the Cam BioBrick vector was successful, another transformation was done into the Cam Biobrick vector, restriction digestions on the previous mini prep from the 1st transformation and 2nd transformation completed, as well as PCR on the mini preps using the corresponding primers for BBa_K11008 and BBa_K110016. In the gel: after the 2 log ladder, the next for lanes have the restriction digestion products (approx 500 ng of plasmid each) and the second four lanes are pcr products in the following order: 08 (1st cam transformation) 16 (1st cam transformation) 08 (2nd transformation) 16 (2nd transformation) for both the restriction digestion and the pcr. The final lane was a control used in the pcr, unrelated to the project. [[Media:090708digestpcr0816.jpg|090708digestpcr0816]] The restriction digestion did not show the insert, while pcr showed a band of the expected size in two of the four samples.

Date: September 2, 2008 Status report by: Allison and Nate Part no.: BBa_K110008 -> BBa_K110016 Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively. Transformation into a Cam BioBrick was completed, and the restriction digestion of the Biobricks from the pGEM vector were checked, comparing the inserts intended to be ligated to  the Kan Biobrick vector and the inserts intended to be ligated to the Cam Biobrick vector; notice that the old inserts are either too faint to see or are not there: [[Media:lanes2thru5OldDigest0816NewDigest0816.jpg|lanes2thru5OldDigest0816NewDigest0816]] Lanes: 1 (2-log ladder), 2 BBa_K110008 old, 3 BBa_K110016 old, 4 BBa_K110008 new, 5 BBa_K110016 new, 6 (2-log ladder) A restriction digestion with EcoRI and PstI was done to check for the insert after ligated and transformed with the Cam Biobrick vector: [[Media:090308digest08_16cam.jpg|090308digest08_16_cam]] Lanes: 1 (old aliquot of 2-log ladder), 2 (new aliquot of 2-log ladder), 3 skip, 4 BBa_K110008, 5 skip, 6 BBa_K110016.

Date: August 19, 2008 Status report by: Allison and Nate Part no.: BBa_K110008 -> BBa_K110016 Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively. Sequences were analyzed. BBa_K110016 had a perfect clone. Since there is not much miniprep left, a transformation was done to generate more clones with the correct sequence. BBa_K110008 had one mutation. Another transformation into a Kan BioBrick vector was attempted but was unsuccessful.

Date: August 12, 2008 Status report by: Allison and Nate Part no.: BBa_K11008 -> BBa_K110016 Part Description: A promoters MFA1 (L+R) and Ste2 (R+L) '''Two more minipreps were completed. DNA concentrations were much higher (>150 ng/ul).'''

Date: August 5, 2008 Status report by: Allison and Nate Part no.: BBa_K110008 -> BBa_K110016 Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively. Sequences were analyzed. BBa_K110016 had a perfect clone. Since there is not much miniprep left, a transformation will be done to generate more clones with the correct sequence. BBa_K110008 had one mutation; additional minipreps from other clones are ready to send off for sequencing, although the DNA concentrations are low. Colonies can be picked and overnight cultures grown for another round of minipreps.

Date: July 29, 2008 Status report by: Allison and Nate Part no.: BBa_K110008 -> BBa_K110016 Part Description: A promoters: MFA1 (L+R) and Ste2 (R+L), respectively. Two samples of mini preps from MFA1 and Ste2 were sent off for sequencing at the end of last week. Results will follow soon.

Date: July 22, 2008 Status report by Allison and Nate Part no.: BBa_K110016 Part Description: Ste2 (R+L) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to   front door; plates at 4 degrees PCR successful? Yes Cloning of PCR product successful: Yes (approx 20 white colonies on one plate) Joining of validated part to adjacent part(s) status: not done Problems to be solved: Current status of this part: 12 mini preps and the restriction digestion were completed; preparing to send 3 samples to be sequenced 

Date: July 22, 2008 Status report by Allison and Nate Part no.: BBa_K110008 Part Description: MFA1 (L+R) Part Location: same as above, plates are at 4 degrees refrigerator near front door PCR successful? Yes (BAD LINK) Cloning of PCR product of successful? Yes (approx 60 white colonies between two plates) Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: Current status of this part: 12 mini preps and the restriction digestion were completed; preparing to send 3 samples to be sequenced [http://www.jhu.edu/iGEM/Group2:MATaSpecificPromoters/2008-7-22.Restriction%20Digests%20of%20BBa_K1100...%2008%20&%2016.Allison%20Suarez%20and%20Nate%20Sotuyo%20.html Restriction Digests of BBa_K1100... 08 & 16]

Date: July 14, 2008 Status report by Allison and Nate Part no.: BBa_K110016 Part Description: Ste2 (R+L) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to  front door; plates at 4 degrees PCR successful? Yes Cloning of PCR product successful: There were many blue colonies (similar to the plate of BB_K110008) Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: Ligation Current status of this part: plates are at 4 degrees; another ligation/transformation will be completed soon

Date: July 14, 2008 Status report by Allison and Nate Part no.: BBa_K110008 Part Description: MFA1 (L+R) Part Location: same as above, plates are at 4 degrees refrigerator near front door PCR successful? Yes Cloning of PCR product of successful? There were mainly light blue colonies (only a couple white colonies) Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: Ligation Current status of this part: plates are at 4 degrees; another ligation/transformation will be completed soon

Date: July 10, 2008 Status report by Allison and Nate Part no.: BBa_K110016 Part Description: Ste2 (R+L) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to front door PCR successful? Yes BioBricks 8 and 16 BioBricks 8 and 16 Using Constant Annealing Short 2Way Stop, Alpha Promoters, & Sapphire FP Cloning of PCR product successful: in progress Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: to be determined Current status of this part: Both PCR protocols (touchdown and second PCR with constant annealing temperature) produced product of the correct size. BBa_K110016 was used as a control in the second PCR with BBa_K110008.

Date: July 10, 2008 Status report by Allison and Nate Part no.: BBa_K110008 Part Description: MFA1 (L+R) Part Location: in a labeled box, second shelf from the top, -20 degrees C refrigerator next to front door PCR successful? Yes BioBricks 8 and 16 BioBricks 8 and 16 Using Constant Annealing Short 2Way Stop, Alpha Promoters, & Sapphire FP Cloning of PCR product successful: in progress Sequencing of cloned PCR product successful: not done Joining of validated part to adjacent part(s) status: not done Problems to be solved: to be determined Current status of this part: PCR was being troubleshooted, appeared to have good results with regular PCR protocol (not touchdown) in which there was a constant annealing temperature of 55 degrees C - see gel