Team:VIT Vellore/Notebook

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NOTEBOOK

The experiments and processes involved

June

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
					1	2
3 Media Preperation Media Preparation × Prepared 100 mL LB broth in 2 flasks each and autoclaved it.	4 Subculture Subculture × E. coli culture was obtained from general lab and thawed at room temperature. 100 μL of the culture was inoculated into the prepared LB broth. Kept in the shaker incubator at 37°C for 24 hours.	5 Streak Streak × 2 LB plates were prepared and autoclaved. The 24 hour culture was streaked on the LB plates. Control was kept blank.	6 Contamination Contamination × Results Contamination was observed in the streaked plates as well as the control plate. The bacteria were overgrown. Inferences The bacteria reached the death phase in 24 hours incubation time. Therefore, incubation time needs to be reduced. The culture obtained from general lab was not pure and hence, could not be used for further experiments.	7 Re-culturing Streak × E. coli culture was obtained from research scholars in Dr. Gothandam’s lab. T2 LB plates were prepared and autoclaved. LB broth (100 mL) was prepared (2 flasks each) and autoclaved. The E. coli culture was thawed at room temperature and 100 μL of it was inoculated in the LB broth. Control was kept blank. The flasks were kept in shaker incubator at 37°C for 20 hours.	8 Streak Streak × The 20 hour culture was streaked on the LB plates. Control was kept blank.	9 Pure Culture Streak × Results Not contamination observed in the streaked plate as well as the control. The culture had grown adequately (no overgrowth). Inferences The bacteria were in the exponential phase of growth in 20 hours incubation time. Therefore, 20 hours incubation is sufficient to maintain viable cells for further experiments. The culture obtained from Dr. Gothandam’s lab was pure and hence could be used for further experiments.
10	11 Reagents Prepared	12 Restriction Digestion Restriction Digestion (works) × Protocol: Restriction Digestion Results Fragments of appropriate size were visualized on the gel. Inferences This indicates the restriction digestion protocol used works successfully. The fragments of appropriate size obtained indicate that the restriction enzymes work successfully.	13 Sub Culture	14	15	16
17 Reagents Prepared Reagents Prepared × Reagents prepared for ligation. Prepared buffers and autoclaved them and other consumables.	18 Ligation Ligation (fail) × Results The band observed in the gel was not of appropriate size. Inferences Ligation failed. Reason (discovered later): Ligase Assay Buffer used for the experiment had been expired.	19 Repeat Ligation Repeat Ligation (works) × Protocol: Ligation Obtained Ligase Assay Buffer from Dr. Gothandam’s lab. Results The band visualized in the gel was of appropriate size. Inferences Ligation done successfully.	20 Sub Culture	21	22	23
24 Reagents Prepared Reagents Prepared × Reagents preparation for PCR. Prepared PCR buffers and autoclaved them.	25 PCR PCR (fail) × Results Bands visualized were not of appropriate size. The bands were not clear and were smeared. The amplicons were not amplified enough and a very faint, thin band was observed. Inferences The dNTP volume added needs to be increased. The agarose concentration in the gel needs to be increased to get a clear separation of the bands.	26 Repeat PCR Repeat PCR (works) × Protocol: PCR Results Bands visualized were of appropriate size. The bands were clear and not smeared. The amplicons were amplified enough and a clear band was observed.	27 Sub Culture	28	29	30

July

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
1	2 Reagents Prepared Reagents Prepared × Reagents preparation for competent cell preparation. Prepared reagents and buffers for competent cell preparation and autoclaved them and other consumables.	3 Make a 24-hour culture.	4 Competent Cell Preperation Competent Cell Preperation × Protocol: Competent Cell Preperation Performed competent cell preparation. Results and Inferences Competent cells were prepared successfully.	5 Sub Culture	6	7
8	9 Reagents Prepared Reagents Prepared × Reagents preparation for transformation. Prepared media (LB and SOC) and autoclaved them along with consumables.	10 Transformation Transformation (fail) × Results No colonies growth was seen on the plate. Inferences Antibiotic concentration could be too high. Transformation could be unsuccessful due to the plausible competent cells to be incompetent for successful transformation. Another possibility could be that the protocol used was not appropriate for transformation.	11 Repeat Transformation Repeat Transformation (works) × Protocol: Transformation Transformation was repeated using a different protocol, with a lower antibiotic concentration. Result Isolated pure E. coli colonies were seen on the plates. No contamination was observed. Inferences Antibiotic concentration used was appropriate. The competent cells prepared in the previous experiment were competent enough for a successful transformation. Protocol used was appropriate for a successful transformation.	12 Sub Culture	13	14
15 Reagents Prepared Reagents Prepared × Reagents preparation for plasmid DNA isolation. Prepared the reagents and buffers for plasmid DNA isolation.	16 Plasmid DNA Isolation Plasmid DNA Isolation (fail) × Results The bands observed were not of appropriate size. Inferences (probable reasons) Concentration of reagents used in buffer preparations was not appropriate. Ethidium bromide was probably not added. Probable mistakes in buffer preparation.	17 Repeat Plasmid DNA Isolation Repeat Plasmid DNA Isolation (fail) × Results The bands observed were not of appropriate size. RNA contamination was observed. Inferences Buffer pH was not appropriate since the buffers used were not freshly prepared. RNAse A was not added.	18 Fresh reagents made Fresh reagents made × Make overnight culture. All reagents and buffers were re-prepared. Culture was thawed at room temperature and inoculated into LB broth and incubated for 20 hours.	19 Repeat Plasmid DNA Isolation Repeat Plasmid DNA Isolation (works) × Protocol: Plasmid DNA Isolation Transformation was repeated using a different protocol, with a lower antibiotic concentration. Results Bands observed were of appropriate size. No RNA contamination was observed. Inferences The composition of the buffers and reagents was amended successfully. The correct size of the bands indicate that the plasmid DNA has been successfully isolated. Absence of contaminating bands indicate that the plasmid DNA obtained is pure and can be used for further characterization.	20 Sub Culture	21
22	23	24	25 Reagents Prepared Reagents Prepared × Reagents preparation for SDS PAGE. Prepared all reagents and buffers for SDS PAGE.	26 SDS Page SDS Page (fail) × Results Experiment failed. Bands were not separated successfully. Inferences (probable reasons) Acrylamide: bis-acrylamide ratio used was inappropriate. Mistakes in reagent and/or buffer preparation.	27 SDS Page Repeat SDS Page (fail) × Results The gel was not destained properly. Inferences Staining and/or destaining time was not correct. (Destaining time was less).	28 Make fresh reagents.
29 SDS Page SDS Page (works) × Protocol: SDS Page Results Bands were separated successfully. Gel was destained successfully. Inferences The protocol used was appropriate. Staining and destaining time was correct. Reagent and buffer preparation was correct.	30 Sub Culture	31 SDS Page (works)

August

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
			1	2	3	4
5 Reagents assembling and autoclaving for alpha amylase experiment.	6 Alpha Amylase Alpha Amylase Experiment × C-source, Organic and inorganic nitrogen. Protocols: Alpha Amylase Characterization Results Relative expression levels of alpha-amylase are compared under different physicochemical conditions.	7 Sub Culture	8	9	10	11
12	13	14	15	16 Replication of restriction digestion (works)	17 Replicate of ligation (works)	18 Replicate of PCR (works)
19 Replicate of competent cell preparation (works)	20 Replicate of electroporation (works)	21 Replicate of PCR (works)	22 Replicate of plasmid DNA isolation (works)	23 Sub Culture	24	25
26 Get ordered construct	27	28	29	30 Sub Culture	31

September

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
						1
2 PCR PCR × Amplification of both the fragments of the construct separately. Results PCR worked successfully. The bands visualized in the gel were of appropriate size. The bands were quite thick, indicating amplification.	3 PCR Clean-up PCR Clean-up × Used commercially available PCR clean-up kit for PCR clean-up. Results Only the amplified DNA is left in the clean-up while the unamplified DNA, leftover dNTPs, MgCl2, etc. is washed up.	4 Nanodrop Nanodrop × Performed to obtain the concentration of the amplified PCR product after PCR clean-up. PCR clean-up reagent was used as the blank. Results The concentration of the PCR amplicon obtained was ~200 μg. Inference PCR amplification is successfully since the concentration of the original DNA template used for PCR was ~2 μg.	5 Sub Culture	6	7	8
9	10	11	12	13	14 Sub Culture	15 Restriction Digestion Restriction Digestion × Restriction digestion was performed of the plasmid backbone and the antisense construct. The Xba I and Spe I sites on both fragments would have sticky ends. Results The fragments visualized on the gel were of appropriate sizes (antisense and backbone). Inference The restriction enzymes worked successfully. The fragments have sticky ends generated for ligation.
16 Ligation Ligation × Ligation performed of the antisense construct with the plasmid backbone. Results The band visualized on the gel was of appropriate size (size of antisense + backbone together). Inferences The fragments were ligated successfully to give a single sequence. This sequence can be transformed further to competent E. coli cells to check for bacterial growth in presence of combination of different antibiotic resistances.	17	18	19	20 Sub Culture	21 Competent Cell Preperation Competent Cell Preperation × Competent cells were prepared and stored at -80°C. Result and Inference The competent cells are capable of transformation.	22 Transformation - Part I Transformation - Part I × Transformation of ampicillin resistant pBR322 in E coli cells. The E. coli competent cells were transformed with ampicillin resistant pBR322 plasmid by heat shock method. The control had non-transformed E. coli competent cells on ampicillin plates. Results The test plate had bacterial colonies growing. No contamination was observed. The control plates had no colonies growing. Inferences Transformation is successful since the test plate shows growth. The plasmid containing ampicillin resistance confers selective pressure to the bacteria to retain the plasmid in ampicillin resistant plates. Non-transformed E. coli cells do not have ampicillin resistance, therefore, there in cell death in control.
23	24	25	26 Transformation - Part II Transformation - Part II × Transformation of our ligated construct into E. coli cells. E. coli competent cells transformed with ligated plasmid construct containing Chloramphenicol resistance by heat shock method. Control had non-transformed E. coli competent cells on chloramphenicol plates. Results The test plate had bacterial colonies growing. No contamination was observed. The control plates had no colonies growing. Inferences Transformation is successful since the test plate shows growth. The plasmid containing chloramphenicol resistance confers selective pressure to the bacteria to retain the plasmid in chloramphenicol resistant plates. Non-transformed E. coli cells do not have chloramphenicol resistance, therefore, there in cell death in control.	27 Sub Culture	28	29 Transformation - Part III Transformation - Part III × Transformation of our construct plasmid (containing chloramphenicol resistance) and pBR322 into E. coli competent cells. Plates contain chloramphenicol and tetracycline. Control had non-transformed E. coli competent cells on Chloramphenicol + Tetracycline plates. Results The test plate had bacterial colonies growing. No contamination was observed. The control plates had no colonies growing. Inferences Transformation is successful since the test plate shows growth. Chloramphenicol + Tetracycline resistance confers selective pressure to the bacteria to retain the plasmid in ampicillin resistant plates. Non-transformed E. coli cells do not have chloramphenicol resistance, therefore, there in cell death in control. The transformed cells from this experiment as referred to as ‘double transformants’.
30

October

Monday	Tuesday	Wednesday	Thursday	Friday	Saturday	Sunday
	1	2	3 Sub Culture	4 Validation Repeat Validation × Plating double transformants containing Chloramphenicol and Tetracycline resistances into Ampicillin plates. Control had non-transformed E. coli competent cells on (Chloramphenicol + Tetracycline + Ampicillin) plates. Results No growth on test plate. No contamination was observed. The control plates had no colonies growing. Inference E. coli cells do not have Chloramphenicol, Tetracycline and Ampicillin resistance natively, therefore control shows no growth. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance and incorporation of pBR322 into the E. coli confers them with Tetracycline and Ampicillin resistance. Therefore, cells transformed with our construct along with pBR322 should survive Chloramphenicol as well as Tetracycline. But, since antisense is also incorporated as a part of our construct which inhibits the Ampicillin resistance gene present in pBR322, thereby resensitizing to the bacteria to Ampicillin. Therefore, the cells do not survive due to Ampicillin present in the plates and the inhibited Ampicillin resistance gene.	5	6
7	8	9	10	11	12	13
14	15	16	17	18	19	20
21	22	23	24	25	26	27
28	29	30	31		28

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