Hyun-Young+L.

=Week 10 & 11 (11/04- 11/15) --= - Spun down second Master plate overnight samples -> Miniprep -> Nanodrop
 * Generally lower yield concentration than first master plate

- Submitted to DNA sequencing using pLIC For and Rev primers -DNA Sequencing from Master Plate 1 ( samples 2-9) and Master Plate 2 came back - Made 20mL of 50mg/mL Kan - Started overnight culture - Protein Expression with positive clone - Small Culture - Found positive controls for the target protein - Made 1000mL of LB media - Protein Expression- Large Culture - Protein Expression - Harvest =Week 9 & 10 (10/21 - 11/01) --=
 * Sample 1 - average of 97.2 ng/uL
 * Sample 2 - average of 77.0 ng/uL
 * Sample 3 - average of 131.8 ng/uL
 * Sample 4 - average of 67.9 ng/uL
 * Sample 5 - average of 71.6 ng/uL
 * Sample 6 - average of 100.1 ng/uL
 * Sample 7 - average of 85.5 ng/uL
 * Sample 8 - average of 82.5 ng/uL
 * Sample 9 - average of 87.5 ng/uL
 * Sample 5 from Master Plate 1 was successful- Both For and Rev results had some missing or non-readable base pairs, but were complimented by each other.
 * 1000mg of Kan sulfate power mixed with 20mL of autoclaved nanopure water and then syringe filtered. Divided into 20 1.7mL tubes and put into -20C fridge.
 * used #5 colony from Master Plate 1
 * 160mL of LB with 160uL of Kan
 * Put into the shaking incubator at 6:20 pm on 11/7/13
 * Performed MidiPrep and got 1000mL of plasmid
 * need to Nanodrop
 * used Sample 5 from Master plate 1 prepared by Miniprep
 * concentration was 83.9 ng/uL -> 0.60uL to get 50ng
 * transformed 50uL of E.coli BL21 (DE3)
 * used Kan plates
 * added 10uL onto Plate A and 50uL to Plate B
 * In the 37 deg Celcius incubator for approx 24hrs and then moved to 4 deg Celcius fridge
 * Plate A seemed to have very tiny colonies but was impossible to pick and isolate each colony -> dispose
 * Plate B had good colonies-> will use for further protein expression steps -> stored in 4 deg Celcius fridge
 * 10g of Trypton and NaCl, 5g of Yeast with 1000mL of water
 * autoclave cycle - LIQUID6
 * When all of 20mL of small culture to 500mL of LB, OD was 0.067, which was much lower than what it should have been, which was 0.1.
 * The large culture put into shaking incubator, since initial OD was lower, will stay in the incubator for longer than normal
 * OD of 0.584 at 9:36PM, Sample 0 was saved; 260uL of 1M IPTG was added to make final concentration of 500uM
 * incubated for 19 hours in water bath shaker at room temp.
 * Forgot to save Sample 1 before centrifugation
 * centrifuged at 5900rpm (approx 6000g) for 20min at 4 degC using JA-10 rotor
 * used pre-made Lysis Buffer (100mM Tris, 300mM NaCl, 10mM Imidazole, pH 7.9)
 * Saved Sample 1 AFTER suspension in Lysis Buffer (don't know if this will be ok)
 * Pellet weight = 3.01g
 * Cell suspension saved in -20degC

Excellent job documenting your progress! Continue working on virtual screening. It'll be helpful for you to make a table of known inhibitors and their structure to be used later on in your final report. - Suman 11/5/13

- Spun down Master plate overnight sample -> pallets formed even though the sample was frozen before spinning - Miniprep on 9 samples -> need to verify the sequence -> DNA sequencing - Nanodrop on 9 samples before DNA sequencing
 * Samples with concentration greater than 100 ng/uL will be submitted twice with For and Rev primers. Those that are less than 100 ng/uL will be submitted with only For primer, and if good sequence comes back, they will be re-submitted with Rev primers.
 * Sample 1- average of 105.9 ng/uL
 * Sample 2 - average of 97.5 ng/uL
 * Sample 3- average of 113.2 ng/uL
 * Sample 4- average of 107.0 ng/uL
 * Sample 5- average of 83.9 ng/uL
 * Sample 6- average of 90.5 ng/uL
 * Sample 7- average of 113.6 ng/uL
 * Sample 8- average of 117.3 ng/uL
 * Sample 9- 74.5 ng/uL

- DNA sequences came back ---> CDS Gene Sequence (codon optimized) missing from targets page-> got it from G.doc-> blast (used tail primers for DNA sequencing ->WRONG!!!! (works but too close to the gene; need to use pLIC primers) -> will pick out good ones and resubmit with pLIC For and Rev) - seq used for Blast: ATGACCGCGACGGCGACCGAAGGTGCGAAACCGCCGTTCGTTTCCCGTAGCGTTCTCGTTACGGGTGGTA ATCGTGGCATTGGTCTGGCTATCGCCCAACGTCTCGCTGCCGACGGTCACAAGGTTGCGGTTACTCACCG TGGTTCTGGTGCGCCGAAAGGTCTGTTCGGTGTTGAGTGCGACGTTACTGATTCTGACGCGGTGGATCGC GCTTTCACCGCTGTTGAAGAACACCAGGGTCCGGTAGAAGTACTCGTATCTAACGCCGGTCTGTCTGCGG ACGCTTTCCTGATGCGTATGACTGAAGAAAAATTCGAAAAAGTTATTAACGCGAACCTGACCGGTGCGTT CCGTGTTGCGCAGCGTGCGTCTCGTTCTATGCAGCGTAACAAGTTTGGTCGTATGATTTTCATCGGTTCT GTTTCTGGTTCTTGGGGCATCGGCAACCAGGCGAATTATGCGGCGAGCAAAGCCGGTGTGATCGGTATGG CGCGTTCCATCGCGCGTGAACTGTCTAAAGCTAATGTTACTGCTAACGTTGTCGCGCCTGGCTACATCGA CACGGATATGACTCGCGCGCTCGACGAGCGTATCCAGCAGGGTGCGCTGCAATTCATCCCGGCCAAACGT GTTGGTACCCCGGCAGAAGTTGCGGGTGTTGTCTCTTTTCTGGCGTCTGAAGACGCATCTTACATTTCCG GTGCCGTTATCCCGGTCGACGGTGGCATGGGTATGGGTCACTAA

--> will resubmit samples 2-9 with pLIC
 * Sample 1: missing a base pair on both For and Rev -> cloning failure confirmed
 * Sample 2: might need to resubmit
 * Sample 3: For result looks good, but Rev result shows few missing or substituted base pairs
 * Sample 4~7,9: only short results came back, not enough to decide success/failure -> will resubmit with pLIC
 * Sample 8: For result too short to decide, Rev result shows missing, substituted, added base pairs

- Resubmitted MtubMabA cloning samples 2 ~ 9 with pLIC For and Rev primers

- Finding positive controls for Virtual Screening : from PubMed papers -> isoniazid, thiolactomycin, thiophenone
 * need to look at Brenda, Binding DB for substrates for MabA/FabG1 in other organisms

- Just in case all of cloning samples fail, made another Master plate
 * 5mL of LB, 5uL of 50 ng/uL Kanamycin per tube
 * total of 9 samples: 3 colonies from each B, C, and D plates

=Week 7 & 8 (10/7 - 10/25) ---= - Cut pNIC - PCR^2 - PCR clean up - Gel check on clean ups Well # -- Content 1 -- skip 2 -- skip 3 -- skip 4 -- skip 5 -- NEB 1k bp DNA ladder 6 -- pNIC vector after clean up 7 -- skip 8 -- Gene Ruler 100 bp DNA ladder 9 -- PCR clean up of MabA from //M. tuberculosis// - Nanodrop on PCR^2 and pNIC accepting vector - Transformation -> 2nd Trial - Transformation Result -> 3 of 4 plates had colonies - Master plate & overnight cultures =Week 5 & 6 (9/23 - 10/4) ---= - To do list: - First trial pNIC cloning failed - nothing grew on 5% sucrose plate - Attempted first trial of pNIC cloning - Nanodropped PCR clean up products
 * Nice work on keeping your page updated. Good captions and analysis of your data. Where are you with virtual? Thank you. -Max 10/21/13
 * pNIC concentration - 38.2 ng/uL
 * increased final volume to 70uL and recalculated other reagents proportionally.
 * made 4 tubes (70uL each)
 * used Primary PCR from summer
 * Made 600uL of Master Mix -> 12 PCR tubes
 * pNIC vector -> combined 4 tubes into one tube and eluted in 50uL
 * PCR^2 -> combined all 12 tubes and eluted in 100uL
 * The bands are in the correct place for both pNIC vector and the insert
 * However, the insert band is very faint (2uL of PCR clean up + 5uL of Loading dye + 13uL of water)
 * Suspicious about the concentration of insert -> will Nanodrop to make sure
 * PCR clean up product of MtubMabA PCR^2 had average concentration of 132.6 ng/uL
 * PCR clean up product of pNIC-Bsa4 accepting vector had average concentration of 124.8 ng/uL
 * __Cohesive end generation__:
 * used NEB Buffer 2 instead of 10X T4 DNA Polymerase Buffer
 * Diluted 1M DTT to 100mM DTT ( 100uL 1M DTT + 900uL water = 1000uL 100mL DTT)
 * the heat block went up as high as 78 deg C (supposed to be at 75 deg C) > will this affect????
 * __Annealing and Transformation :__
 * Made 4 tubes (vector : insert)
 * A- 2:4
 * B- 2:5
 * C- 3:8
 * D- 2:10
 * used E.coli DH5alpha (Lot # C2987H)
 * Colonies were big ( colonies were not this big when I grew up pNIC plasmid in DH5alpha) -> made sure that I used DH5alpha and NOT BL21
 * Do not know why the colonies are so big.
 * Number of colonies:
 * A: 0
 * B: 20
 * C: 23
 * D: 15
 * Ratio of 3 vector: 8 insert yielded highest number of colonies
 * Will make master plate
 * Picked 3 colonies from each B, C, D --> 9 colonies on the master plate and made 9 overnight culture.
 * grown overnight
 * stored overnight culture in -20degC BEFORE spinning down --> if pallets form, ok: if not, need to grow overnight cultures again using master plate.
 * NIice Job with the captions and analyses. Keep up the good work. -Max 10/07/2013
 * cut pNIC + PCR^2 (make bunch)
 * pNIC might have been too old (grown up during summer)
 * Extended RT incubation during cohesive end generation might have had effect
 * heat shock too long??? (30sec according to protocol)
 * Maybe need to grow up pNIC again
 * During cohesive end generation: incubated at RT for 1hr instead of 30min
 * had only 5X T4 DNA polymerase Buffer instead of 10X -> doubled the buffer vol and decreased water volume
 * used E. coli DH5alpha
 * heat shock for 45 seconds
 * pNIC vector - grown up during summer, sequence verified
 * A- 2 vector : 4 insert
 * B- 3 vector : 5 insert
 * MabA from M. tuberculosis prepared on 9/3/13 - avg. concentration of 197.2 ng/uL
 * MabA from M. tuberculosis prepared on 7/18/13 - avg. concentration of 21.6 ng/uL
 * Dr.B said it's too old -> will dispose
 * Dr.B said it's too old -> will dispose

- PCR clean up on PCR^2 and pNIC accepting vector Well # -- Content 1 -- NEB 100 bp DNA ladder 2 -- PCR^2 clean up of MabA from M. tuberculosis Why are these different heights on the gel? - Dr. B 100113 3 -- PCR^2 clean up from summer (eluted in 500uL by accident) <-- 4 -- skip 5 -- NEB 1k bp DNA ladder 6 -- pNIC vector clean up 7 -- pNIC vector clean up from summer
 * pNIC vector Nanodrop result after SacB gene cut out and after PCR clean up ; prepared on 09/30/13
 * average concentration of 100.6 ng/uL
 * pNIC vector Nanodrop result after SacB gene cut out and after PCR clean up ; prepared on 07/19/13
 * average concentration of 23.7 ng/uL
 * also too old -> will dispose
 * also too old -> will dispose
 * PCR^2: eluted in 50uL
 * pNIC: eluted in 30uL
 * ran a gel check along with summer PCR^ clean up (eluted in 500uL instead) and summer pNIC vector clean up just to see how they come out

- Gel check on pNIC accepting vector Well # -- Content 1 -- NEB 1kb bp DNA ladder 2 -- skip 3 -- Tube A: pNIC-Bsa4 with SacB gene cut out 4 -- skip 5 -- Tube B: pNIC-Bsa4 with SacB gene cut out 6 -- skip - Made 50X TAE buffer - Nanodropped PCR^2 from secondary PCR (summer) =Week 3 & 4 (9/9 - 9/20)= Young, ok making some progress but keep the pressure on to get your clonign done. For gel images - embed the image vs. linking it. - Dr. B 100113
 * I made two tubes of accepting vector just in case, hence Tube A and B.
 * pNIC plasmid's concentration (prepared by Midiprep) was too low, so I increased the final volume to 60uL and calculated other reagents' volume proportionally
 * The upper bands in Lane 3&5 are the big section of pNic plasmid without the SacB gene and the lower bands are the SacB gene DNA that have been cut out. The upper bands are darker than the lower bands even though their concentration are the same, because smaller SacB gene has much less space for EtBr to bind, which is the reagent that makes DNA glow.
 * The two separate (upper and lower) bands indicate that pNIC has been cut correctly.
 * 57.1 mL glacier acetic acid
 * 242 g Tris Base
 * 37.2 g Na2EDTA H20
 * Added those ingredients to 1000 mL graduated cylinder and filled it up to 1000 mL mark with nanopure water.
 * average concentration of 304.6 ng/uL

- messed up PCR clean up at the end of summer, restarting from oligo mix - Made Oligo mix (little pieces of my gene MtubMabA), Primary PCR (gluing the pieces together), Secondary PCR (copying only full length genes), PCR^2 (amplifying secondary PCR product) - (9/13) Attempted gel electrophoresis on Primary, Secondary, and PCR^2. Voltage set to 130, ran about 50 min. When finished, the buffer inside the rig was hotter than usual and the gel was extremely fragile. It was impossible to take the gel out of the buffer without breaking the gel. - (9/16) Re-attempted gel electrophoresis on Secondary and PCR^2. Voltage set at 110 and ran for 50 min. Run was successful. Well # -- Content 1 -- NEB 100 bp DNA ladder 2 -- Secondary PCR using Primary PCR from 9/9/13 3 -- PCR^2 using Secondary PCR (9/9/13) 4 -- skip 5 -- NEB 100 bp DNA ladder 6 -- Secondary PCR using Primary PCR from Summer 7 -- PCR^2 using Secondary PCR (Summer) - pNIC-Bsa4 vector preparation. My pNIC concentration was 42.2 ng/uL. In order to get 2.25 ug (according to the protocol) I needed 53.3 uL, which was too much volume than the protocol (total of 25uL). So, I readjusted the total volume to be 60uL and calculated proportions of other reagents. After combining all the necessary reagents, the tubes were put into 37 degree Celcius water bath from 2:14 PM to 4:25 PM. Then the tubes were stored at -20 C for later use. (PCR clean up, gel electrophoresis, and Nanodrop will be performed later.) =Week 1 & 2 (8/28 - 9/6)= Young - show some images for results (e.g. at least an image of PyMol refresher. Should have some PCR attempts here. WOrk on making pNIC-Bsa4 also. - Dr. B 090913
 * At next attempt, I will lower the voltage, run the gel for longer time, and leave the gel in the casket when putting into the rig.
 * The bands are between 700 and 800 bp, which is the correct spot, since my gene is 744bp. On Well #3, there is a big smear between 100 bp and 200 bp. This might be contamination or gene fragments from Primary and Secondary PCR.

__8/30__ - made 1000mL of LB (no antibiotics) __9/3__ - PyMol refresher = = = = = = =+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++= =Week8 (7/22 -7/26)=

__7/22__ - research presentation - DNA sequence not back yet =**Week7 (7/15 - 7/19)-**= Young - good job. Band looks nice. Be sure to do teh LIC cloning exercise (paper based). I would recommend growting more pNIC-Bsa4 since the conc was low. Be sure to elute in only 500ul - Dr. B 071713

__7/19__ - PCR^2 clean up ERROR: eluted in 500uL instead of 50uL.... also did not Nanodrop it before clean up - Vector preparation ERROR: did not run the gel before clean up -> too little sample for running gel - 3rd MidiPrep - submitted to DNA sequencing with pLIC-for and rev
 * solutions to both errors: will redo both steps using 3rd MidiPrep product
 * collected pellets from yesterday's culture and cont. to MidiPrep
 * eluted with only 500uL of elution buffer instead of 1000uL to increase the concentration
 * average concentration of 42.2 ng/uL
 * halved the elution the volume but concentration was not much higher

__7/18__ - PCR clean up -> stored in my box (-20C) - Vector preparation - 160mL of pNIC Bsa4 overnight culture for MidiPrep
 * had 200uL of PCR^2 product -> needed 1000uL of Binding Soln.... did not fit into the column
 * divided into two columns: 500uL of Binding soln and 100uL of PCR^2 in each column
 * used Tris-HCl (0.5M, pH of 6.92) instead of Elution soln provided in the clean up box
 * used 37.5 ng/uL pNIC-Bsa4 (prepared by first MidiPrep on 6/11 because second MidiPrepped pNIC conc. was so low ->25.6 ng/uL)
 * doubled the final volume from 25uL to 50uL due to low plasmid concentration... needed 60uL for 2.25ug of pNIC
 * water bath for 3 hrs -> will due clean up tomorrow, stored in -20C

__7/17__ - after successful secondary PCR, proceed to PCR squared - PCR result - Ran gel electrophoresis of primary and secondary
 * massive producing of secondary PCR product
 * much will be lost during clean up process
 * well# -- content
 * 1 5uL of 1k bp DNA ladder
 * 2 Primary PCR of MtubMabA
 * 3 Secondary PCR of MtubMabA
 * Primary should be a smear and secondary should have a band
 * used 1k bp ladder
 * added 2uL of Blue Juice to 15uL of samples

__7/16__ - Primary and Secondary PCR on MtubMabA - Made Oligo mix for MtubMabA - DNA seq of pNIC-Bsa4 prepared by MidiPrep came back code NNNNNNNNNNNNNNNNNNNNNNNNNNNANNNNNANATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTG GGTACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATATACCTGCCGTTCACTATTATTTAGTGAAATGA GATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAA TGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGA GGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGAT AAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTT ATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGA CATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAACCTTTACTACCGCACTGCTGGCAGGAGGCGCA ACTCAAGCGTTTGCGAAAGAAACGAACCAAAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATAT GCTGCAAATCCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAAAAATATCTCTT CTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGACGGCACTGTCGCAAACTATCACGGCTACCAC ATCGTCTTTGCATTAGCCGGANATCCTAAAAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAAC TTCTATTGACAGCTNGNAAAACGCTGGNCNNGTCNTTAANACAGCGACAANTCGATGCNNTGATNCTATCCTAAANANCN ANNNCNAGAATGGNCNGGNTCANCNNNNTTTANNTNNNGACGNAAAATCGTTNNNTCTACNNNGATTNNNCNGGNAANNN NCGNNNNAANNCTGANANTNNNNNNNTNNCNNNTCNNCNTCNNNNNNNTCNTTNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNANNNTACGNNNNNNTNNNANNNNNNNNNNNNNNNNNNNNNNGNNN code code NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCG AANNCNNNTCCGTATCCACCTTTACTGGAGACCGTCAATGCCAATNNNNNNNCNGCATTTTCTTTTGCGTTTTTATTTGT TAACTGTTAATTGTCCTTGTTCAAGGATGCTGTCTTTGACAACNNATGTTTTCTTGCCTTTGATGTTCAGCAGGAAGCTA GGCGCAAACGTTGATTGTTTGTCTGCGTAGAATCCTCTGTTTGTCATATAGCTTGTAATCACGACATTGTTTCCTTTCGC TTGAGGTACAGCGAAGTGTGAGTAAGTAAAGGTTACATCGTTAGGATCAAGATCCATTTTTAACACAAGGCCAGTTTTGT TCAGCGGCTTGTATGGGCCAGTTAAAGAATTAGAAACATAACCAAGCATGTAAATATCGTTAGACGTAATGCCGTCAATC GTCATTTTTGATCCGCGGGAGTCAGTGAACAGGTACCATTTGCCGTTCATTTTAAAGACGTTCGCGCGTTCAATTTCATC TGTTACTGTGTTAGATGCAATCAGCGGTTTCATCACTTTTTTCAGTGTGTAATCATCGTTTAGCTCAATCATACCGAGAG CGCCGTTTGCTAACTCAGCCGTGCGTTTTTTATCGCTTTGCAGAAGTTTTTGACTTTCTTGACGGAAGAATGATGTGCTT TTGCCNTANNATGCTTTGTTAAATAAAGATTCTTCGCCTTGGTAGNCATCTTCAGTTCCAGTGTTTGCNNCAAATANNAA GTATTTGTGGCCTTTATCTNCTACNTAGTGAGGNNNNNCTCANNNANGGNNGNCGCCTGANNNGNANTNGCCNTCATCNN NGAACTGCTGTACNNATTNANACNNNNTNNCGNNACCNTNNNNNN
 * with pLIC-For
 * pLIC-For Blast result
 * with pLIC - Rev

code __7/15__ - made Oligo Mix for my target -> MtubMabA =**Week6 (7/8 - 7/12)-**=
 * pLIC-Rev Blast result
 * have 18 primers
 * final concentration of 1uM

agarose gel electrophoresis result of restriction enzyme digest.
 * -** RE Digest
 * needs DNA seq Analysis (virtual gel) for further analysis

- Nanodropped for concentration -> avg of 25.6 ng/uL
 * -** pNIC-Bsa4 Midi Prep

=**Week5 (7/1 - 7/5)---**=

- pmCherry PCR =**Week4 (6/24 - 6/28)**=
 * well# -- content
 * 1 --- 100bp DNA ladder
 * 2 --- 0.01 ng/uL pmCherry DNA with VDSR primer
 * 3 --- 0.1 ng/uL pmCherry DNA with VDSR primer
 * 4 --- 1.0 ng/uL pmCherry DNA with VDSR primer
 * 5 --- no DNA with VDSR (could be contamination or trans-over from well 4)
 * 6 --- 0.01 ng/uL pmCherry DNA with M13 primer
 * 7 --- 0.1 ng/uL pmCherry DNA with M13 primer
 * 8 --- 1.0 ng/uL pmCherry DNA with M13 primer
 * 9 --- no DNA with M13 primer (could be contamination or trans- over from well 8)
 * ** VDSR primers worked better than M13 on pmCherry. The marks were much brighter and distinctive.

--SICK =**Week3 (6/17 - 6/21)---**=

- 2nd trial of pGBR22 PCR - tried to run gel on 2nd PCR
 * used pGBR22 with concentration of 156.9 ng/uL
 * ERROR: did NOT dilute taq
 * wells were broken due to weak (really soft) gel and samples were lost in the buffer

__ 6/19 __

- pGFP gel result -> failed...no bands... only smears - pGFP (green) PCR - overnight culture growth of yopH in pNIC-Bsa4
 * Well# -- content
 * 1 - 100bp DNA ladder
 * 2 - 0.05 ng/uL pGFP plasmid with VDSR primers
 * 3 - 0.5 ng/uL pGFP plasmid with VDSR primers (LOST!!!!!!!!!)
 * 4 - 4.5 ng/uL pGFP plasmid with VDSR primers
 * 5 - no plasmid with VDSR primers
 * 6 - 0.05 ng/uL pGFP plasmid with M13 primers
 * 7 - 0.5 ng/uL pGFP plasmid with M13 primers
 * 8 - 4.5 ng/uL pGFP plasmid with M13 primers
 * 9 - no plasmid with M13primers
 * ** including final extension period to repeating cycle may have affected the result
 * plasmid concentration of 450 ng/uL
 * for primers, used M13-F&R and VDS1&2
 * ERROR: 'Final Extension' step in PCR was included in repeating cycle (wasn't suppose to)
 * samples left on the PCR machine overnight and collected to my box on 6/20
 * saved samples for running gel later
 * 100mL of LB + 100uL of Kan

__6/20__ - yopH Protein Expression - pmCherry (red) PCR
 * did not do transformation (putting plasmid into the bacteria), instead used someone else's already-transformed colony
 * monitored OD (optical density)
 * final: 20mL of overnight culture solution to 500mL of LB to make OD measurement to 0.1
 * incubated in shaking incubator for 2 hours to make OD 0.533
 * Sample 0: collected 500uL of soln (cell lysate before induction)
 * added IPTG (250uL) & incubated for 4 hrs
 * Sample 1: collected 500uL of soln after incubation (cell lysate after induction)
 * harvested by centrifugation
 * pellet weight of 0.13g
 * plasmid concentration of 112.6 ng/uL
 * primers: VDSR and M13
 * did not make master mix because the protocol did not have any measurements

=**Week2 (6/10 - 6/14)**=

- result of 1st PCR
 * well#--- content
 * 1 100 bp DNA ladder
 * 2-5-- Grace T.'s sample A-D
 * 6-9-- My sample A-D -> PCR failed

__6/10__ - Overnight culture using colonies from Wk1 (pNIC Bsa4)

__6/11__ - Midi Prep using sample from 6/10 (pNIC Bsa4) - Nanodropped resulting plasmid --- avg. concentration of 37.5ng/uL

- Submitted DNA seq using pLic-For as primer

To determine the 'N's, I viewed Chromatogram from coreweb.utexas.edu and checked each 'N' up to Query 801. Did not check after base 801 since the result toward the end is relatively unreliable since only used pLic-For. For improved result, could resubmit using pLic-Rev

=**Week1 (6/3 - 6/7)-**= __**6/3**__ - Dilution of primer: sp6 promoter from 100 microM to 10 microM - Nanodrop of pmCherry

used 204ng/uL pmCherry with M13F primer 1st trial: said they will rerun with more samples 2nd trial: came back with four Ns- could have been error in prepping the sample or used wrong primer __6/4__ - Made 5 agar plates with kanamycin
 * -** Submitted DNA sequencing:

__6/5__ - 1st PCR

=__6/6- 6/7__= =- Transformation Efficiency= Plate A: 1080 colonies / 2ng plasmid = 540 colonies per ng plasmid (pNIC BSA4) Plate B: 456 colonies / 10ng plasmid = 45.6 colonies per ng plasmid (pNIC BSA4) Plate C: 68 colonies / 50ng plasmid = 1.36 colonies per ng plasmid (pNIC BSA4)
 * Transformation Efficiency calculation:**