Brianna+G.+(RP+Vet)

=**Week 14, 15**= Thursday 12/4/2014
 * pNIC was digested. Due to time, digested pNIC was not run through a gel yet to verify if digestion worked. Will run gel later.
 * cohesive end generation was performed, treating digested pNIC and kpblaGES-5 with T4 polymerase to create sticky ends.
 * Annealing and transformation performed, ligating kpblagES-5 with digested pNIC, and then transforming the ligation mix into DH5alpha cells.
 * the following ratios were used: Tube A (6 gene : 1 vector), Tube B (7 gene : 1 vector)
 * Expressed cells in 100uL of SOC media in 37C shaking incubator for 1 hr. Then spread expression onto Kan-Suc plates and incubated again at 37C (no shaking) for 1.5 days
 * The cloning plates are shown in figures 24 and 25. Bacterial colonies were produced, indicating potential success of positive clone. A master plate and further expression and DNA extraction (mini prep) with sequencing will be conducted in January.


 * Figure 24.** LIC-G plate A (6:1) after 1.5 days incubation(12/6) of 37C incubation. All of the yellow specs are bacterial colonies.


 * Figure 25.** LIC-G plate B (7:1) after 1.5 days (12/6) of 37C incubation. All of the yellow specs are bacterial colonies.

Tuesday 11/25/2014 ==
 * PCR clean up was performed on PCR2-J into one tube and PCR2-K into another tube. The concentrations were as follows:
 * Figure 22.** Nanodrop result of kpblaGES-5-J that was PCR cleaned-up shown as 10mm Absorbance vs wavelength nm. The concentration was measured 136.4ng/uL.

**Figure 23.** Nanodrop result of kpblaGES-5-K that was PCR cleaned-up shown as 10mm Absorbance vs wavelength nm. The concentration was measured as 112.3ng/uL.
These completed kpblaGES-5 genes are now ready for independent ligation cloning.

=**Week 11, 12, 13**= Wednesday 11/19/2014
 * PCR2-K performed to further amplify kpblaGES-5 and have extra gene for multiple rounds of cloning, using 2PCR-M2, GES5 For- and rev- primers-D, and thermoprogram with annealing temperature at 55.5C and annealing and elongation time at 30 sec. Performed 2 rounds (8 tubes).
 * both tubes produced bands around 864bp (figure 21), indicating success for PCR2-K! Any smudges below 500bp is hoped to be discarded during PCR clean up.

__Figure 21.__ Agarose gel of 1kb DNA ladder in lane 1, PCR2-K1 in lane 2, and PCR2-K5 in lane 3.

Tuesday 11/18/2014 __Figure 20.__ Agarose gel of 1kb DNA ladder in lane 1, PCR2-J1 in lane 2, and PCR2-K5 in lane 3.
 * PCR2-J performed to further amplify kpblaGES-5, using 2PCR-M1, GES5 For- and rev- primers-D, and thermoprogram with annealing temperature at 55.5C and annealing and elongation time at 30 sec. Performed 2 rounds (8 tubes).
 * both tubes produced bands around 864bp (figure 20), indicating success for PCR2-J! And further verification of success for 2PCR-M! Any smudges below 500bp is hoped to be discarded during PCR clean up.

Monday 11/17/2014
 * 1PCR-M made to make template for 2PCR, using oligomix-F and a thermoprogram with annealing temperature at 55.5C and annealing and elongation time at 30 sec.
 * large smudge appeared (lane 7 in figure 19), covering from below 500bp marker and up to 8000bp marker. Lots of the gene was produced! but kpblaGES-5 is only 864bp, so for everything above the 1000bp marker, it's hoped for that it will be broken up to only make the 864bp gene in 2PCR.

__Figure 19.__ Agarose gel of 1kb DNA ladder in lane 1, Saul's PCR2 in lanes 2-5, nothing in land 6, and 1PCR-M in lane 7.

__Figure 18__. Agarose gel of 1kb DNA ladder in lane 1, 2PCR-M1 in lane2, 2PCR-M2 in lane 3, 2PCR-M3 in lane 4, 2PCR-M4 in lane 5.
 * 2PCR-M made to assemble kpblaGES-5 gene, to serve as a template for PCR2. Used 1PCR-M as template and a thermoprogram with annealing temperature at 55.5C and annealing and elongation time at 30 sec. 4 rounds of 2PCR-M were performed (i.e. 4 tubes).
 * The gel was totally prepared wrong because room temperature 1x TAE was added to a post-microwaved (hot-warm) 1xTAE, 1% agarose, and EtBr, cartridge -- did not think 70mL of 1xTAE were used to make the solution in the cartridge -- so it solidified upon pouring the extra 1x TAE, thus distorting the gel.
 * as a result, the bands and 1kb DNA ladder looked very distorted and with lots of smudging! But bands can kind of be made out, and it seems like if the gel was made properly, then distinct bands would have resulted. Because of time and not wanting to waste more resources, it is assumed a success, and further verification can be made at PCR2 anyways

Friday 11/14/2014 __Figure 17.__ Agarose gel of 1kb DNA ladder in lane 1 and 1PCR-L in lane 2.
 * 1PCR-L made to make template for 2PCR since the 1PCR-K is probably already denatured and 2PCR needs to be remade anyways to perform PCR2. Used oligomix-E and thermoprogram where annealing temperature was 55.5C and annealing and elongation time were 30 sec.
 * no smudge appeared for 1PCR-L (figure 17). This may have been because the wrong/old oligomix was used on accident -- should be using oligomix-F. Will fix that for next 1PCR.

Tuesday 11/4/2014 __Figure 16.__ Agarose gel of 1kb DNA ladder in lane 1, PCR2-I1 in lane 2, PCR2-I2 in lane 3, PCR2-I3 in lane 4, PCR2-I4 in lane 5
 * 1PCR-I for further amplification of kpblaGES-5 gene was performed using 2PCR-L1, kpblaGES-5 for- and rev- primers-D, and thermoprogram where annealing temperature was 55.5C and annealing and elongation time were 30 sec.
 * no bands appeared for PCR2-I (figure 16) -- not sur why this is. Since this procedure has already failed before using 2PCR-L1, will remake another template for PCR2

=**Week 8, 9, and 10**= Saturday 11/1/2014 __Figure 15.__ Agarose gel of 1kb DNA ladder in lane 1, and 2PCR-H tubes 1-4 samples across lanes 2-5 respectively.
 * PCR2-H for further amplification of kpblaGES-5 gene was performed using 2PCR-L1, kpGES-5 for- and rev- primers-D, and thermoprogram where annealing temperature was 55.5C and annealing and elongation time were 30sec.
 * No bands appeared for PCR2-H, only smudges below 500bp (figure 15) -- not sure why this happened, will redo PCR2 with same procedures and ingredients

Wednesday 10/29/2014
 * prepared kpGES-5 for- and rev- primers-D from newly ordered kpGES-5 stocks

Monday 10/27/2014 __Figure 14.__ Agarose gel of 1kb DNA ladder in lane 1, 2PCR-L1 in lane 2, and 2PCR-L2 in lane 3.
 * Agarose gel of 2PCR-L1 and L2 ran
 * Bands appeared for both 2PCR-L1 and L2 around 864bp with some streaks above the band and a smudge for 2PCR-L2 under 500bp marker (figure 14)

Thursday 10/23/2014
 * Prepared kpGES-5 for- and rev- primers-C (using summer 2014 stock since newly ordered primers had not arrived yet)
 * 2PCR-L1 and L2 was performed using 1PCR-K and thermoprogram where annealing temperature was 55.5C and annealing and elongation time was lengthened to 30sec. L1 used for- and rev- primers-C. L2 used 1st oligo and last oligo to serve as primers.
 * agarose gel for 2PCR-L1 and L2 was ran Monday10/27/2014

Wednesday 10/22/2014 __Figure 13.__ Agarose gel of 1kb DNA ladder in lane 1 and 2PCR-K in lane 2.
 * 2PCR-K for amplifying kpblaGES-5 gene was performed using 1PCR-K, for- and rev- primers-B (prepared 9/1/2014 from summer 2014 stock) and a thermoprogram where annealing temperature was lowered to 55.5C and annealing and elongation time was lengthened to 30sec
 * very faint band appeared under 1000bp (kpblaGES-5 864bp; figure 13) -- 2PCR-K not very successful, need new primers that aren't denatured.

Wednesday 10/15/2014 __Figure 12.__ Agarose gel of 1kb DNA ladder in lane 1 and 2PCR-J in lane 2.
 * 2PCR-J for amplifying kpblaGES-5 gene was performed using 1PCR-K and the standard the thermoprogram as directed by protocol (annealing temp 72C and annealing and elongation time 10sec and 20sec respectively)
 * smudge appeared below 500bp (figure 12) -- some segments of kpblaGES-5 amplified but in general, 2PCR-J failed -- need to use thermoprogram with lowered annealing temp to 55C and annealing and elongation time lengthened to 30sec, similar to that used in 1PCR-K

Monday 10/13/2014 __Figure 11.__ Agarose gel of 1kb DNA ladder in lane 1 and 1PCR-K in lane 2.
 * Oligomix-F of kpblaGES5 was made from Fall 14B (newly ordered) to perform 1PCR
 * 1PCR-K for assembling kpblaGES-5 gene was performed using oligomix-F and a thermoprogram where annealing temperature was lowered to 55.5C and annealing and elongation time was lengthened to 30sec
 * large bright streak (figure 11) indicated that 1PCR-K was a success

Wednesday 10/8/2014
 * Week 7 (10/6/2014 - 10/10/2014)**
 * Performed 2PCR-I (made from 1PCR-J3) using "l amborghini" PCR machine (all other ingredients taken out and prepared fresh from stock, for- and rev- primers made 9/1/2014), assuming 1PCR-J worked. Resulting sample would be ran through a gel (figure 10). This 2PCR-J3 was performed with 1PCR-J3 as the DNA template hoping a complete kpblaGES5 will form and amplify -- standard 2PCR procedures conducted.
 * Agarose gel showed no bands for 2PCR-I (figure 10) --> 2PCR-I failed. The 1 kb DNA ladder had slightly smeared bands and the 100bp DNA ladder came out completely smeared when it was supposed to have distinct bands. With this being the case, the gel may have to be re-run with the 2PCR-I sample, in order to verify if 2PCR-I really did not work, or if the gel was just not prepared right.

__Figure 10.__ Agarose gel of 1kb DNA ladder in lane 1, 100bp DNA ladder in lane 2, and 2PCR-I in lane 3. Jairo's 2PCR was in lane 5, and Gio's 2PCR was in lane 7. The 100bp DNA ladder showed a smear and there were no bands in lanes 3, 5, and 7.

Monday 10/6/2014
 * Made an agarose gel for 1PCR-J samples. After running the samples, the lanes showed no bands (figure 9) --> all 1PCR-J failed to make a complete kpblaGES-5 gene. There was an intense, thick smudge for 1PCR-J3, below the 500bp marker, indicating that some oligos amplified and may have assembled but not all of the oligos assembled all together. A 2PCR may be performed using 1PCR-J3 just in case kpblaGES-5 assembles completely in the 2PCR. Also with a smudge thick and intense, the oligomix-E may be missing an oligo so a new oligomix should be made and retried for 1PCR with optimized conditions (lower 55.5C annealing and 30 sec annealing and elongation).

__Figure 9.__ Agarose gel of 1kb DNA ladder in lane 1, 1PCR-J1 in lane 2, 1PCR-J2 in lane 3, 1PCR-J3 in lane 4, and 1PCR-J4 in lane 5. There are extremely faint small smudges below the 500bp marker in lanes 2,3, and 5. There is a distinct, thick smudge in lane 4 (1PCR-J3) below the 500bp marker.

Friday 10/3/2014
 * Week 6 (9/29/2014 - 10/3/2014)**
 * Performed 1PCR-J (4 sets using oligmix-E, following a procedure with a 30sec annealing, 30sec extension, and an annealing temperature gradient -- 58C, 57.2C, 55.5C, 54.2C) using "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock. Resulting sample was ran in 1% agarose gel (figure 9).

Wednesday 10/1/2014
 * Performed 1PCR-H (standard procedure using oligomix-E) and 1PCR-I (elongated annealing time 10sec-> 30sec and elongated extension time 20sec-> 30sec using oligomix-E) using "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock). Resulting sample was ran in 1% agarose gel (figure 8).
 * Agarose gel for 1PCR-H and 1PCR-I showed no bands (figure 8) --> 1PCR-H and 1PCR-I failed. There are however very slight smudges below the 500bp but that may be a few amplified segments of the gene, not assembled together. To optimize the PCR procedure further, in addition to time lengthening, is lower temperature to esure that the oligos assemble with one another.

__Figure 8.__ Agarose gel of 1kb DNA ladder in lane 1, 1PCR-H in lane 2, and 1PCR-I in lane 3. There are no bands in lanes 2 and 3 but there are faint smudges below the 500bp marker.

Tuesday 9/30/2014
 * Performed 2PCR-H (made from 1PCR-G) using "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock, for- and rev- primers made 9/1/2014), assuming 1PCR-G worked. Resulting sample would be ran with 1PCR-G sample (figure 7)
 * Agarose gel for 1PCR-G and 2PCR-H showed no bands (figure 7) --> 1PCR-G and 2PCR-H failed. 2PCR-H obviously failed because there was no template from 1PCR-G. It was very unexpected that 1PCR-G did not work considering every ingredient used was fresh, including the NEW OLIGOS. In order to improve chances of success, 1PCR will be performed again using oligomix-E, where annealing time would be lengthened and temperature would be decreased -- optimizing conditions.

__ Figure 7. __ Agarose gel of a 1kb DNA ladder in lane 1, 1PCR-G sample in lane 2, 2PCR-H sample in lane 3, and 100bp ladder in lane 4. No bands evident in lanes 2 and 3.

Thursday 9/25/2014
 * Week 5 (9/22/2014 - 9/26/2014)**
 * Performed 1PCR-G (made from oligomix-E) using "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock). Resulting sample would be ran in 1% agarose gel with 2PCR-H (figure 7).

Tuesday 9/23/2014
 * Prepared oligomix-E; made from newly ordered oligo box (Fall14B)

Thursday 9/18/2014
 * Week 4 (9/15/2014 - 9/19/2014)**
 * Ran 1st run for virtual screening refresher

Friday 9/12/2014
 * Week 3 (9/7/2014 - 9/13/2014)**
 * Made a 1% agarose gel and ran 2PCR-C (made from summer 14 and used in fall for PCR2 but failed - to verify if these samples were still good), 2PCR-G, and 1PCR-F (figure 6). No bands for 1PCR-F samples, indicating that oligos might be denatured and new ones need to be ordered (had already all other components: Q5, dNTP, and PCR machine - none were the problem). No bands for 2PCR-G samples, indicating that there was no assembled kpblaGES-5 to begin with in the 1PCR-E samples. Bands in 2PCR-C indicating that the kpblaGES-5 gene is still intact/assembled however since this sample did not produce more kpblaGES-5 for PCR2, the ends of the gene might be denatured or the primers-for adn -rev might be denatured - should order new primers-for and -rev just in case.

__Figure 6.__ Agarose gel of 1kb DNA ladder in lane 1, 2PCR-C2 in lane 2, 2PCR-C3 in lane 3, 2PCR-G1 in lane 4, 2PCR-G2 in lane 5, 1PCR-F1 in lane 6, 1PCR-F2 in lane 7, and 1kb DNA ladder in lane 8. Two distinct bands in lanes 2 and 3 (for 2PCR-C) around 800bp with some smudges below the 500bp. Lanes 4-7 all have smudges below the 500bp mark.

Thursday 9/11/2014
 * Worked on PyMol refresher (finished)
 * Performed 1PCR-F1 (made from oligomix-C) and 1PCR-F2 (made from oligomix-D) using "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock)
 * Performed 2PCR-G1 (made from 1PCR-E1) and 2PCR-G2 (made from 1PCR-E2) using "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock) - performed just incase the smudge below the 1000bp marker in the 1PCR-E gel was assembled kpblaGES-5.

Wednesday 9/10/2014 __Figure 5.__ Agarose gel of 1kb DNA ladder in lane 1, pNIC PCR Q5-A in lane 2, pNIC PCR Q5-B in lane 3, pNIC PCR Q5-D in lane 4, pNIC PCR Q5-F in lane 5, pNIC PCR Q5-G in lane 6. Distinct, thick, intense bands in lanes 2-6, all slightly above 2000bp.
 * Worked on PyMol refresher (not yet finished)
 * Made a 1% agarose gel and ran PCR samples of pNIC. (figure 5) Gel showed distinct, thick, and intense bands for all pNICs treated with different Q5 polymerases (lanes 2-6). This indicates that PCR worked with all the Q5 polymerases tested and the dNTP; the problem for the recent PCRs on kpblaGES5 thus lies in the kpblaGES5 gene. The oligos from stock may be denatured or an oligo might be missing in oligomix-B. A new oligomix will be made and re-tried for PCR. If the PCR for the new oligomix works, then the project can proceed in motion to 2PCR (the problem was just an oligo was missing from the mix). If the PCR for the new oligomix fails, then a new oligo set needs to be ordered because the stock oligos had denatured.
 * Made two new oligomixes for kpblaGES5 from thawed sum14B box -- oligomix-C (made by me) and oligomix-D (made by Grace).

Tuesday 9/9/2014
 * Began PyMol refresher (not yet finished)
 * Prepared Q5 polymerase (used 5 different Q5 tubes, labeled A, B, D, F, and G; Q5 tubes C and E did not have enough volume)
 * Separated master mix into 5 separate tubes and added the diluted pNIC from yesterday and each tube had a different Q5 polymerase added.
 * "lamborghini" PCR machine used with following thermoprogram: lid 98C, vol 25uL
 * 98C 30sec initial denaturation
 * 98C 10sec denaturation
 * 50C 30 sec annealing
 * 72C 1min elongation
 * repeat steps 2-4 35 times (35x cycles)
 * 72C 2min final elongation
 * 4C forever storage
 * stored pNIC PCR samples in -20C freezer

Monday 9/8/2014
 * Prepared master mix for PCR of pNIC28-Bsa4 using pLIC-for and pLIC-rev primers. pNIC used is from 6/19/14.
 * Diluted pNIC prepared to 0.819 ng/uL

Saturday 9/6/2014 __Figure 4.__ Agarose gel of 1kb DNA ladder in lane 1, 1PCR-E1 in lane 2, 1PCR-E2 in lane 3, 1kb DNA ladder in lane 4, PCR2-G1 in lane 5, PCR2-G2 in lane 6, PCR2-G3 in lane 7, and PCR2-G4 in lane 8. No distinct bands in lanes 2-3 nor in lanes 5-8. Light streaks under 500bp in lanes 2-3 and intense streaks under 500bp in lanes 5-8.
 * Week 2 (8/31/2014 - 9/6/2014)**
 * Performed PCR2-G using 2PCR-C3 (made in 7/2014 - might still be in good condition for a PCR2 procedure) and "ferrari" PCR machine (all other ingredients taken out and prepared fresh from stock)
 * Performed 1PCR-E (1 rounds) using oligomix-B adn "ferrari" PCR machine (all other ingredients taken out and prepared fresh from stock)
 * *Accident* - cancelled 1PCR-E when done and accidentally cancelled PCR2-G while it was still running (about 8 min left); looked at machine for options for 5min then implemented the following program: lid 98C, vol 50uL
 * 98C 1sec initial denaturation
 * 98C 20sec denaturation
 * 58C 10sec annealing
 * 72C 20sec elongation
 * repeat steps 2-4 8 times (8x cycles)
 * 72C 2min final elongation
 * 4C forever storage
 * Agarose gel showed no bands for 1PCR-E nor for PCR2-G (figure 4) --> 1PCR-E and PCR2-G failed; probably not PCR machine since "lamborghini" and "ferrari had already been used for the PCR procedures. Fail might be because DNA was denatured (oligomix-B and/or 2PCR-C and/or kpblaGES5-for-B and -rev-B), Q5 polymerase was denatured, or dNTP denatured. (contaminated buffer as a cause is unlikely so it will not be considered for now)

Wednesday 9/3/2014
 * Performed 1PCR-D of kpblaGES5 using oligomix-B and "lamborghini" machine (all other ingredients taken out and prepared fresh from stock)
 * Performed 2PCR-E of kpblaGES5 using 1PCR-D and "lamborghini" machine (all other ingredients taken out and prepared fresh from stock)
 * Agarose gel showed not bands for 1PCR-D nor for 2PCR-E (figure 3) --> 1PCR-D and 2PCR-E failed; might be because the "lamborghini" PCR machine is not working, might be that the stock from which the oligomix-B was prepared was denatured, might be that the oligomix was made incorrectly, might be that the dNTP or the Q5 polymerase had denatured.

__Figure 3.__ Agarose gel of 1kb DNA ladder in lane 1, 1PCR-D in lane 2, and 2PCR-F in lane 3. No bands visible in lanes 2 and 3.

Tuesday 9/2/2014 __Figure 2.__ Agarose gel of 1kb DNA ladder in lane 1, 1PCR-C in lane 2, and 2PCR-E in lane 3. No bands visible in lanes 2 and 3.
 * Agarose gel showed no bands for 1PCR-C nor for 2PCR-D (figure 2) --> 1PCR-B and 2PCR-D failed; might be because the 2mM dNTP were not fresh and may have denatured, therefore not allowing proper amplification

Monday 9/1/2014
 * Prepared oligo mix for kpblaGES5 from thawed sum14B box (labeled oligomix-B)
 * Prepared 20uM kpblaGES-for primer and 20uM kpblaGES5-rev primer from their respective stocks (opened and ordered from summer 2014)
 * Performed 1PCR-C of kpblaGES5 using oligomix-B (freshly made that day), 2mM dNTP (prepared 7/2014), and "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock)
 * Performed 2PCR-E of kpblaGES5 using 1PCR-C, 2mM dNTP (prepared 7/2014), 20uM kpblaGES5-for-B primer (freshly made that day), 20uM kpblaGES5-rev-B (freshly made that day), and "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock)
 * Stored 1PCR-C and 2PCR-E products in -20C

Wednesday 8/28/2014 __Figure 1.__ Agarose gel of 1kb DNA ladder in lane 1, 1PCR-B in lane 2, and 2PCR-D in lane 3. No bands visible in lanes 2 and 3.
 * Week 1 (8/25/2014 - 8/30/2014)**
 * Performed 1PCR-B of kpblaGES5 using oligomix-A (prepared 7/1/14), 2mM dNTP (prepared 7/2014), and "lamborghini" PCR machine (all other ingredients taken out fresh and prepared from stock)
 * Performed 2PCR-D of kpblaGES5 using 1PCR-B, 2mM dNTP (prepared 7/2014), 20uM kpblaGES5-for primer-A (prepared 7/8/2014), 20uM kpblaGES5-rev-A primer (prepared 7/8/2014), and "lamborghini" PCR machine (all other ingredients taken out and prepared fresh from stock)
 * Agarose gel showed no bands for 1PCR-B nor for 2PCR-D (figure 1) --> 1PCR-B and 2PCR-D failed; might be because oligomix-A (possible kpblaGES5-for-A and -rev-A primers too) were denatured, therefore not allowing proper amplification