Team:Yale/Our Project/Notebook/Week 3

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lab notebook: week 3 (6/21-6/27)

• Monday 6/21--Miniprep Biobrick plasmids from overnight culture, but a mix up samples, necessitating a diagnostic digest.
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• Miniprepped the cultures grown overnight according to standard microcentrifuge protocol, but mixed up tubes so hope to sort them out with a diagnostic double digest
• The similar size and identical backgrounds of the Biobricks makes many of them difficult to distinguish by restriction digest failing cut sites within the Biobricks themselves. EcoRI and PstI are available but B0015, C0012, and J23114 (all 35 bp in around 3 kb backgrounds) are too hard to tell apart by such a digestion. However, a EcoRI/PstI digest should be able to tell them from R0011 and pSB74--the former will be divided into 1.1 kb and 2.1 kb fragments while the latter will end up as 3.6 kb and 9.4 kb fragments.
• Before setting up digestion, used nanodrop to determine the concentration of the ten unknown samples (two of each plasmid)

Sample	1	2	3	4	5	6	7	8	9	10
DNA Concentration (ng/ul)	22.0	76.1	51.5	15.5	98.7	303.0	27.9	11.4	82.6	306.6

• Samples 2,4, and 7 were too dilute to be of use, but ran 50 uL digestions of the other seven using 1 ng worth of DNA,0.5 uL of 100x BSA, 5 uL of NEB EcoRI 10xBuffer, 0.5 uL of NEB EcoRI, 0.5 uL of NEB PstI, and enough water to round out the volume. These digestions were put in the thermocycler for 30 minutes at 37˚C
• Ran 10 uL of each digestion reaction with 2 uL loading buffer versus a 1 kb ladder in a 0.8% agarose gel stained with EtBr. The resulting image is shown below, with columns containing the ladder, sample 2, sample 3, sample 5, sample 6, sample 8, sample 9, and sample 10 from left to right.
• Samples 3 and 10 match the fragment sizes expected for R0011, while 5 might be pSB74, though the fragment size seems rather large. The other assignments are rather ambiguous, so gave up on identifying tubes.
• Redid inoculation of liquid cultures of strains containing pSB74, R0011, J23114, C0012, and B0015 and left in ampicillin LB on the shaker overnight.
This day's activities are also recorded on pages 18 and 19 of the hard copy lab notebook.
• Tuesday 6/22--Miniprep redos
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• Began miniprep of overnight cultures, but in initial cell pelleting phase the centrifuge spun the tops of the tubes, spilling them.
• Started over with the inoculation of two 5 mL liquid ampicillin LB cultures each of pSB74, R0011, C0012,J23114, and B0015 containing strains.
• When OD was approximately 0.6, took 0.5 mL of each solution and mixed with 0.5 mL of 50% filter-sterilized glycerol and set aside for glycerol stocks.
• Let cultures continue to grow all day, then miniprepped via the microcentrifuge protocol, eluting into 40 uL of water.
This day's activities are also recorded on page 18 of the hard copy lab notebook.
• Wednesday 6/23--Making TSI agar slants & transformation of P0312
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Nanodrop of 6/22 miniprep
Nanodropped the ten minipreps from the day before and got the following results:
Sample	J23114a	J23114b	C0012a	C0012b	R0011a	R0011b	B0015a	B0015b	pSB74a	pSB74b
DNA Concentration (ng/ul)	122.9	91.3	17.4	57.9/td>	86.8	59.5	70.2	83.7	67.3	44.2
Also flash froze in liquid N₂ the glycerol DNA solutions from 6/22 and put them in the -80˚C freezer.
Triple Sugar Iron (TSI) agar preparation
So that can readily determine whether bacteria are producing hydrogen sulfide, created a batch of TSI agar slants according to the following recipe , with ampicillin as the added antibiotic and tubes of the medium allowed to solidify on an angle to produce a characteristic slant. If a culture containing thiosulfate reductase is grown in such a slant, it will turn the medium black by precipitating out black iron sulfide, an obvious visual cue of hydrogen sulfide production.
Transformation of BBa_P0312
After realizing that Biobrick P0312 can be used in place of C0012 and R0011, transformed LE392 with P0312 according to the standard transformation protocol and plated on ampicillin LB plates to grow overnight in the incubator at 37˚C. (Because no Amp LB plates were prepared, simply spread the antibiotic topically on a plain LB plate prior to adding transformants.) This day's labwork is also recorded on pages 18, 20, and 21 of the hard copy lab notebook.
• Thursday 6/24--primer ordering & IPTG-inducible promoter(P0312) transformants
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• Finally ordered the primers designed on 6/15 as well as standard VF2 and VR primers for Biobricks.
• Saw that P0312 transformants had grown, though apparently ampicillin was not thoroughly distributed on the plate, as there was lawn growth at the very edges and scattered colonies in the middle. Used one of these middle resistant colonies to inoculate a 5 mLliquid ampicillin LB culture to grow overnight on the shaker at 37˚C for miniprep the next day.
This day's labwork is also recorded on page 22 of the hard copy lab notebook.
• Friday 6/25--P0312 miniprep, pSB74 transform, & pouring plates
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• Miniprepped the overnight culture of P0312 in LE392 according to the standard microcentrifuge protocol.
• Transformed BL21, LE392, and DH5alpha with pSB74 according to this standard transformation protocol
  and plated on ampicillin LB plates to incubate overnight at 37˚C.
• Prepared 1 liter's worth of ampicillin LB plates and 500 mL of plain LB plates according to this recipe. The ampicillin 1000x stock solution was made by dissolving 2.0 g of ampicillin in 20.0 mL of water, filter-sterilizing the resultant solution, and storing it in 1 mL aliquots in the freezer. Incubated five of the plain LB plates overnight as a sterility check.
This day's activities are also recorded on page 22 of the hard copy lab notebook.