Difference between revisions of "Team:IISER Pune/Notebook"
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<a href="#Chromoproteins"><li>Detection Chromoproteins Notebook </li> </a> | <a href="#Chromoproteins"><li>Detection Chromoproteins Notebook </li> </a> | ||
<a href=".pdf"><li>Detection Carotenoids Notebook (pdf)</li> </a> | <a href=".pdf"><li>Detection Carotenoids Notebook (pdf)</li> </a> | ||
+ | <a href="#Enzyme_Substrate"><li> Detection Enzyme Substrate Notebook</li></a> | ||
+ | <a href=".pdf"><li>Termination notebook(pdf)</li> </a> | ||
+ | |||
+ | |||
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− | <div id=EnzymeSubstrate> | + | <div id="EnzymeSubstrate"> |
<a name="Enzyme_Substrate"></a> | <a name="Enzyme_Substrate"></a> |
Revision as of 01:21, 19 September 2015
List of Note Books
- Hijack Module (Fast robust oscillator) Notebook
- Detection Chromoproteins Notebook
- Detection Carotenoids Notebook (pdf)
- Detection Enzyme Substrate Notebook
- Termination notebook(pdf)
Hijack Module (Fast robust oscillator) Notebook
25th June 2015
DH5α competent cells were transformed with the following biobricks:
BioBrick |
Part |
Backbone |
Kit plate, Well |
BBa_C0080 |
araC protein coding sequence |
pSB1C3 |
3,11B |
BBa_C0012 |
LacI protein coding sequence |
pSB1C3 |
pSB1C3 |
The plates were kept at 37°C for 14 hours.
26th June 2015
Transformation results:
Biobrick |
Number of colonies |
BBa_C0080 |
0 |
BBa_C0012 |
3 |
As number of colonies obtained was very low, the transformation of these biobricks was repeated.
27th June 2015
Transformation results:
Biobrick |
Number of colonies |
BBa_C0080 |
3 |
BBa_C0012 |
60 |
Inoculation:
A colony from BBa_C0080 plate was inoculated in 7ml LB media + 7μL Chloramphenicol. Same was done for BBa_C0012. These cultures were kept at 37°C for 12 hours.
28th June 2015
No growth was observed in both the liquid cultures.
A new colony was inoculated again.
29th June 2015
Growth observed in the cultures of BBa_C0080 and BBa_C0012. Gylcerol stocks were made for both.
Plasmid isolation was done using Alkaline Lysis protocol for the 2 cultures. (1.5ml culture x 3)
Concentrations obtained from nanodrop:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_C0080 (1) |
3301.8 |
2.02 |
BBa_C0080 (2) |
3670.9 |
1.98 |
BBa_C0080 (3) |
2777.6 |
1.95 |
BBa_C0012 (1) |
4560.0 |
1.97 |
BBa_C0012 (2) |
4542.1 |
1.94 |
BBa_C0012 (3) |
5123.5 |
1.61 |
A single digest was set for one of each of these plasmids:
(all volumes in μL) |
BBa_C0012 |
BBa_C0080 |
PstI |
0.3 |
0.3 |
10x NEBuffer 3 |
1 |
1 |
100x BSA |
0.1 |
0.1 |
DNA |
0.5 |
0.5 |
MilliQ H20 |
8.1 |
8.1 |
Total Volume |
10 |
10 |
- Kept at 37°C for 2 hours
- Heat Inactivation: 80°C for 20 minutes
Gel Electrophoresis:
- Digested samples run on a 1% gel.
- Voltage: 100V
- Time run: 50 minutes
Both samples showed an RNA smear.
This occurred as the Alkaline Lysis solution I did
not have RNAse. After observing this smear, RNAse was added to the solution.
Re-inoculation of the two cultures was done
1st July 2015
A plasmid isolation using Alkaline lysis was done.
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_C0080 (1) |
4548.1 |
1.98 |
BBa_C0080 (2) |
3411.9 |
2.01 |
BBa_C0080 (3) |
3492.6 |
1.98 |
BBa_C0012 (1) |
2258.4 |
1.99 |
BBa_C0012 (2) |
2442.6 |
2.00 |
BBa_C0012 (3) |
2691.4 |
1.97 |
A digest was set up with a sample of each plasmid and then run on a gel.
The gel showed bands at correct length, but the band intensity was extremely low and did not reflect the concentration obtained on nanodrop. A new method for plasmid isolation was needed.
2nd July 2015
BBa_K094120 arrived from iGEM Headquarters and was inoculated in 5ml LB media + 5μL Ampicillin. Kept at 37°C for 12 hours.
3rd July 2015
The liquid culture of BBa_K094120 was plated and kept overnight at 37°C
5th July 2015
A single colony was inoculated for BBa_K094120. Kept at 37°C for 12 hours
6th July 2015
Plasmid isolation of BBa_K094120 using Alkaline Lysis:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_K094120 (1) |
1050.8 |
1.88 |
BBa_K094120(2) |
1493.7 |
1.95 |
BBa_K094120 (3) |
1365.2 |
1.97 |
29th July 2015
1% gel run with all samples for the 3 plasmids BBa_C0080, BBa_C0012, BBa_K094120.
All bands seen had low intensity which did not correlate to the concentration on nanodrop.
11th August 2015
Revive glycerol stocks for BBa_C0080, BBa_K094120 and BBa_C0012. A 50 ml culture was inoculated for BBa_C0080 and BBa_C0012 for a Midiprep using Qiagen kit.
Kept at 37°C overnight
12th August 2015
Midiprep using Qiagen Kit results for BBa_C0080 and BBa_C0012:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_C0012 |
7.0 |
3.62 |
BBa_C0080 |
27.8 |
2.07 |
Since results obtained were very low, a new “Qiagen spin miniprep” kit was obtained.
17th August 2015
Primary cultures used to make an overnight 5ml culture of BBa_C0080, BBa_C0012 and BBa_K094120.
18th August 2015
Qiagen Spin Miniprep protocol followed. Results:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_K094120 |
250.7 |
1.91 |
BBa_C0080 |
165.5 |
1.92 |
BBa_C0012 |
195.4 |
1.92 |
A digestion was set to check plasmid length:
(all volumes in μL) |
BBa_C0080 |
BBa_C0012 |
BBa_K094120 |
DNA |
2 |
2 |
2 |
EcoRI |
0.2 |
0.2 |
0.2 |
10x EcoRI Buffer |
1 |
1 |
1 |
100x BSA |
0.1 |
0.1 |
0.1 |
MilliQ H20 |
6.4 |
6.4 |
6.4 |
Total Volume |
10 |
10 |
10 |
Gel: 1% gel run at 100V for 40 minutes.
The bands obtained for all 3 plasmids were at the correct length. The uncut plasmid ran faster than cut plasmid.
21st August 2015
Inoculated BBa_I13504 (GFP, used as reporter in oscillator construct) from previously transformed plate. Kept at 37°C for 12 hours
22nd August 2015
- Miniprep of BBa_I13504:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_I13504 |
122.4 |
1.94 |
- Transformation of the biobrick BBa_B0034 (RBS) in DH5α competent cells (Ampicillin antibiotic)
- Plates kept at 37°C for 14 hours
23rd August 2015
Inoculated a colony for BBa_B0034 and kept at 37°C for 12 hours.
24th August 2015
Plasmid Isolation using Qiagen Miniprep kit:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_B0034 |
95.2 |
1.95 |
25th August 2015
- Transformation of the biobrick BBa_B0015 (double terminator) in DH5α competent cells (Chloramphenicol antibiotic)
- Plates kept at 37°C for 14 hours
26th August 2015
Transformation Result:
Number of colonies for BBa_B0015 = 8
- Inoculated BBa_B0015 in 5ml LB media + 5 μL Chloramphenicol
- Kept at 37°C for 12 hours
28th August 2015
Plasmid Isolation of BBa_B0015 using Qiagen Miniprep kit:
Sample |
Concentration (ng/μL) |
A260/A280 |
BBa_B0034 |
72.3 |
1.95 |
29th August 2015
Single Digest to check plasmid length:
(all volumes in μL) |
BBa_B0015 |
BBa_I13504 |
BBa_B0034 |
DNA |
4 |
2 |
2 |
EcoRI |
0.2 |
0.2 |
0.2 |
10x EcoRI Buffer |
1 |
1 |
1 |
100x BSA |
0.1 |
0.1 |
0.1 |
MilliQ H20 |
5.7 |
5.7 |
5.7 |
Total Volume |
10 |
10 |
10 |
Gel: 1% gel run with uncut and cut samples at 100V for 40 minutes.
The 3 single cut bands were at the right length.
6th September 2015
Digestion for 3A assembly of constructs of the oscillator construct:
(all volumes given are in μL) |
BBa_ K094120 |
BBa_ C0080 |
BBa_ C0012 |
BBa_ I13504 |
BBa_ B0034 |
BBa_ B0015 |
Ampicillin Backbone |
Chloramphenicol Backbone |
Kanamycin Backbone |
DNA |
3 |
3 |
3 |
3 |
4 |
5 |
10 |
10 |
10 |
10x NEBuffer 2 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
2.5 |
BSA |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
EcoRI |
0.5 |
0.5 |
0.5 |
- |
- |
- |
0.5 |
0.5 |
0.5 |
XbaI |
- |
- |
- |
0.5 |
0.5 |
0.5 |
- |
- |
- |
SpeI |
0.5 |
0.5 |
0.5 |
- |
- |
- |
- |
- |
- |
PstI |
- |
- |
- |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
H20 |
13 |
13 |
13 |
13 |
12 |
11 |
6 |
6 |
6 |
Total |
20 |
20 |
20 |
20 |
20 |
20 |
20 |
20 |
20 |
- Kept at 37°C for 5 hours
- Heat Inactivation at 80°C for 20 minutes
- The following devices were to be assembled:
- BBa_K094120 + BBa_B0034 + Chloramphenicol backbone = Device 2a
- BBa_C0080 + BBa_B0015 + Kanamycin backbone = Device 2b
- BBa_C0012 + + BBa_B0015 + Ampicillin backbone = Device 2c
- BBa_K094120 + BBa_I13504 + Kanamycin backbone = Device 2d
Ligation:
(all volumes in μL) |
Device 2a |
Device 2b |
Device 2c |
Device 2d |
Ampicillin Backbone |
- |
- |
2 |
- |
Chloramphenicol Backbone |
2 |
- |
- |
- |
Kanamycin Backbone |
- |
2 |
- |
2 |
BBa_K094120 |
2 |
- |
- |
2 |
BBa_B0034 |
2 |
- |
- |
- |
BBa_C0080 |
- |
2 |
- |
- |
BBa_B0015 |
- |
2 |
2 |
- |
BBa_C0012 |
- |
- |
2 |
- |
BBa_I13504 |
- |
- |
- |
2 |
T4 DNA ligase |
0.5 |
0.5 |
0.5 |
0.5 |
Ligase Buffer |
1 |
1 |
1 |
1 |
MilliQ H20 |
2.5 |
2.5 |
2.5 |
2.5 |
Total Volume |
20 |
20 |
20 |
20 |
Ligation kept overnight at room temperature.
7th September 2015
- Transformation of the ligation mix into DH5α cells
- Kept at 37°C overnight
8th September 2015
Transformation results:
Ampicillin Negative Control : Lawn growth
Kanamycin Negative Control : Zero colonies
Plate |
Number of Colonies |
2a |
10 |
2b |
12 |
2c |
Lawn growth |
2d |
5 |
Ampicillin degraded. 2c re-transformed
Inoculation of a single colony from each plate in 5ml LB media + 5μL antibiotic
Kept at 37°C for 12 hours
9th September 2015
Miniprep by Qiagen Kit:
Sample |
Concentration (ng/μL) |
A260/A280 |
2a |
283.4 |
1.91 |
2b |
122.3 |
2.09 |
2d |
226.6 |
1.96 |
Single Digest with EcoRI to check plasmid length:
Backbone-Backbone ligation was observed on the gel.
A new ligation was setup. This time insert:vector ratio was increased. Kept at 37°C for 2 hours.
These ligation mix were transformed into Dh5α competent cells.
10th September 2015
Growth observed in negative control plates.
An overnight digestion was set for the individual plasmids again. Kept at 37°C for 12 hours
11th September 2015
Ligation set for 2 hours and then transformed.
12th September 2015
Colonies of device 2a, 2b and 2c obtained. No colonies on 2d.
3 colonies from each plate were inoculated, incubated for 12 hours at 37°C and miniprepped.
These were then digested with EcoRI to check the length of the plasmid on a gel.
The bands were as expected.
An overnight digest followed by 2 hour ligation was set to assemble to following device:
Device |
Part1 |
Part2 |
Backbone |
5a |
2a |
2b |
pSB1A3 |
5b |
2a |
2c |
pSB1K3 |
Two samples of 5b were assembled – one low copy and second high copy backbone
BBa_I13504 in Ampicillin backbone was chosen to assemble device 2d. This biobrick was digested and ligated.
Devices 5a, 5b and 2d were transformed.
13th Spetember 2015
Transformation Results:
Negative Control: Zero
Plate |
Number of Colonies |
5a |
15 |
5b (low copy) |
70 |
5b (high copy) |
26 |
2d |
10 |
Three colonies were inoculated from each plate, incubated at 37°C and then miniprepped.
These were then single digested using EcorI to check on gel.
2d, 5a and 5b high copy showed correct bands.
18th September 2015
2d construct was characterized by induction with different concentrations of IPTG and arabinose.
Chromoproteins
8th June, 2015
● The following biobrick parts were revived from the iGEM 2015 distribution plates using the iGEM protocol Standard protocol.
● Transformation was carried with Ultra-competent cells for DH5α strain of E.coli (with 200µL of LB).
● 200µL of the revived product was plated on LA+antibiotic.
● The plates were kept at 37℃ overnight (12-16 hours).
Part Number |
Location in Kit |
Backbone |
J23110 (Constitutive Promoter) |
Plate 4, Well 19D |
Ampicillin |
K592025 (RBS+amilCP) |
Plate 1, Well 19C |
Chloramphenicol |
J04450 (RFP) |
Plate 4, Well 2H |
Ampicillin |
Negative Control: For both antibiotics (Amp and Cam)
Positive Control: RFP
9th June, 2015
Part Number |
Number of colonies |
J23110 (Promoter) |
Many colonies |
K592025 (RBS+amilCP) |
Many single colonies |
J04450 (RFP) |
Lawn growth |
Negative Control |
No colonies |
● Next, the colonies were inoculated in 5mL LB+ 0.1% antibiotic.
● They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.
10th June, 2015
● Growth was observed in K592025, but no growth in J23110.
● Again a colony was picked from the plate with J23110 and inoculated in 5mL LB+ 0.1% antibiotic.
● They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.
11th June, 2015
● Again no growth was observed in J23110 (even after inoculating twice).
● Transformation was done again for J23110
● Different promoter was also transformed.
Part Number |
Location in Kit |
Backbone |
J23119 (Constitutive Promoter) |
Plate 3, Well 17O |
Chloramphenicol |
13th June, 2015
● The two promoters were tranformed with 200µL SOC media which was prepared using the iGEM protocol (earlier transformations with LB).
● 200µL of the revived product was plated on LA+antibiotic.
● The plates were kept at 37℃ overnight (12-16 hours).
14th June, 2015
Part Number |
Number of colonies |
J23110 (Promoter 1) |
30 |
K592025 (RBS+amilCP) |
3 |
J23119 (Promoter 2) |
47 |
Negative Control |
No colonies |
● The colonies obtained were inoculated in 5mL LB+ 0.1% antibiotic.
● They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.
18th June, 2015
Plasmid isolation was carried out using the Alkaline Lysis Protocol from Sambrook and Maniatis.
Resuspended in TE Buffer.
Results of Nanodrop:
Sample |
Vial Number |
A260/A280 |
Concentration (ng/µL) |
K592025 |
1 |
1.99 |
6691.9 |
|
2 |
2.11 |
4749.5 |
|
3 |
2.12 |
4158.3 |
J23119 |
1 |
2.10 |
4865.3 |
|
2 |
2.09 |
2757.4 |
|
3 |
2.10 |
4902.3 |
J23110 |
1 |
2.09 |
5121.8 |
|
2 |
2.15 |
8148.0 |
|
3 |
2.13 |
3223.8 |
● The alkaline lysis buffer I wasn’t added with RNase.
● The plasmid isolation was repeated with RNase added to Buffer I.
27th June, 2015
● The colonies were again inoculated in 5mL LB+ 0.1% antibiotic.
● They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.
28th June, 2015
● RNase (10mg/mL stock) was added to Alkaline Lysis Buffer I.
● Resuspension in milliQ water.
Results of Nanodrop:
Sample |
Vial Number |
A260/A280 |
Concentration (ng/µL) |
K592025 |
1 |
2.13 |
56.9 |
|
2 |
1.39 |
67.4 |
|
3 |
2.06 |
58.5 |
J23119 |
1 |
2.16 |
34.1 |
|
2 |
2.15 |
32.3 |
|
3 |
1.39 |
116.1 |
J23110 |
1 |
2.08 |
158.4 |
|
2 |
2.02 |
3222.1 |
|
3 |
1.95 |
580.4 |
29th June, 2015
● The colonies were again inoculated in 5mL LB+ 0.1% antibiotic.
● They were kept in an incubator at 37℃ overnight (12-16 hours) at around 180-200 rpm.
30th June-10th July, 2015
Again, alkaline lysis was done.
Results of Nanodrop:
Date |
Sample |
Vial Number |
A260/A280 |
Concentration (ng/µL) |
30/6 |
J23119 |
1 |
1.94 |
2376.8 |
30/6 |
J23119 |
2 |
1.95 |
2301.2 |
30/6 |
J23119 |
3 |
1.88 |
1662.9 |
8/7 |
J23119 |
1 |
1.99 |
1616.5 |
8/7 |
J23110 |
2 |
1.98 |
618.9 |
10/7 |
K592025 |
1 |
1.96 |
1641.1 |
10/7 |
K592025 |
2 |
1.97 |
1457.0 |
10/7 |
K592025 |
3 |
1.99 |
1404.6 |
|
|
|
|
26th August and 3rd September
Results for Nanodrop by Qiagen spin mini kit
Sample |
A260/A280 |
Concentration (ng/μL) |
J23119 |
2.04 |
121.1 |
J23119 |
1.89 |
329.3 |
K592029 |
1.96 |
158 |
K592029 |
1.88 |
407.3 |
26th August
Single digestion reaction
(in μL) |
K592025 |
J23119 |
EcoRI |
0.3 |
0.3 |
DNA |
2 |
2 |
10XEcoRI Buffer |
1 |
1 |
100X BSA |
0.1 |
0.1 |
Distilled water |
6.6 |
6.6 |
Total Volume |
10 |
10 |
27th August
3A assembly double digestion
(in μL) |
Promoter(J23119) |
amilCP(K592025) |
Linear backbone |
DNA |
2 |
2 |
10 |
10X Buffer2 |
2.5 |
2.5 |
2.5 |
100X BSA |
0.5 |
0.5 |
0.5 |
EcoRI |
0.5 |
- |
0.5 |
SpeI |
0.5 |
- |
- |
PstI |
- |
0.5 |
0.5 |
XbaI |
- |
0.5 |
- |
DpnI |
- |
- |
0.5 |
Distlled water |
14 |
14 |
5.5 |
Total Volume |
20 |
20 |
20 |
Kept at 37°C for 4 hours, heat-kill at 80°C for 20min.
Colonies obtained (~10), were inoculated and miniprep was done with qiagen kit.
Table3: Double digestion for 2A assembly |
2A assembly Double digestion:
(in μL) |
Promoter(J23119) |
amilCP(K592025) |
DNA |
7.8 |
7 |
10X Buffer2 |
1 |
1 |
100X BSA |
0.2 |
0.2 |
SpeI |
0.5 |
0.5 |
PstI |
0.5 |
- |
XbaI |
- |
0.5 |
Distilled water |
0 |
0.8 |
Total Volume |
10 |
10 |
Detection Enzyme Substrate Notebook
DETECTION: Enzyme substrate reaction
Parts:
Biobrick | Part | Size(bp) | Location | |
1 | BBa_K1509003 | Constitutive promoter | 2129 | Ordered from iGEM HQ |
2 | BBa_K823016 | RBS + full length LacZ | 5163 | Kit plate 2 |
5/08/15
Transformation of BBa_K823016 (LacZ gene along RBS) plasmid in DH5α competent cells
Protocol: iGEM Transformation protocol
Master plate is made
6/08/15
Observed colonies on the plate
12/08/15
Liquid culture is prepared for BBa_K823016
Protocol
- 5ml of LB in 15ml falcon
- Add 5µl of cam
- Pick a colony from the plate and dip in falcon
- Incubate for 12-14hrs
22/08/15
BBa_K1509003 (promoter) is ordered and got from iGEM HQ. NEB10 is the cell line
Preparation of liquid culture
- 5ml LB in 15ml falcon
- Add 5µl of cam
- A loop of nichrome dipped in NEB10 cells containing promoter plasmid
- Incubate for 12-14hrs
23/08/15
Streaking of plate
- Prepare a cam media plate of 20ml LA
- Streak the plate using previously prepared liquid culture
- Overnight Incubation
24/08/15
Grow liquid stalk culture
- 5ml LB in 15ml falcon
- Add 5µl of cam
- Pick a colony from master plate and dip in media
- Overnight incubation
Preparation of glycerol stalk
- 0.5ml 80% glycerol in cryotube
- 0.5ml bacterial culture
- Flash freeze it using liquid nitrogen
24/08/15
Plasmid isolation by alkaline lysis
Chemicals required:
- CAM antibiotic
- LB media
- Alkaline lysis buffer 1
- Alkaline lysis buffer 2
- Alkaline lysis buffer 3
- Ethanol
- Phenol: Chloroform (1:1)
- STE
- TE with RNase A
Protocol
- Inoculate 5ml of rich media containing the appropriate antibiotic (CAM)
- Incubate culture overnight at 37 deg
- Pour 5ml of culture into a microfuge tube
- Centrifuge in microfuge for 3 min at 4deg
- Remove media by aspiration leaving bacterial pellet as dry as possible
- Resuspend the pellet in 100microl of ice-cold alkaline lysis solution 1 by vortexing
- Add 200microl of alkaline lysis solution 2 , mix with inverting
- Add 150 microl ice-cold alkaline lysis solution 3 mix with invertexing
- Store tube on ice for 3-5 min
- Centrifuge bacterial lysate ,5 min 13000rpm at 4deg in microfuge
- Transfer supernatant to fresh tube
- Add equal volume of phenol: chloroform
- Mix the organic and aqueous phase by vortexing
- Centrifuge at maximum speed 2 min at 4deg
- Transfer the aqueous upper layer to fresh tube
- Add 2 volume of ethanol
- Mix by vortexing for 2 min
- Centrifuge for 5min at 4deg
- Remove supernatant by aspiration
- Remove all liquid keeping inverted position
- Add 1ml of ethanol to pellet
- Centrifuge for 2min at 4deg
- Remove all supernatant by aspiration
- Remove ethanol beads and evaporate it at room temperature
- Dissolve nucleic acid in 50microl of TE containing RNases A
- Vortex the solution gently for 2 sec
- Store DNA solution at -20deg
Result: no DNA pellet after precipitation by ethanol
Comment: Most probably step 12 is done in incorrect way
Inoculation of a colony of BBa_K823016 and BBa_K1509003
25/08/15
Plasmid isolation of BBa_K1509003 using QIAGEN miniprep kit
Protocol: QIAGEN Miniprep gravity column is used
Result: No DNA after precipitation by isopropanol addition
26/08/15
Inoculation of BBa_K823016 and BBa_K1509003
Plasmid isolation using QIAGEN Miniprep spin column
Protocol: QIAGEN Miniprep spin column protocol is used
Results:
part | Concentration(ng/µl) |
BBa_K823016 | 52.2 |
BBa_K1509003 | 38.7 |
Inoculation of BBa_K823016 and BBa_K1509003 for alkaline lysis
27/08/15
Plasmid isolation using Alkaline lysis Miniprep in AB Lab
Chemicals Required: Alkaline lysis buffer 1, 2, 3 buffer 2 freshly prepared, RNases
Protocol: standard protocol is used from sambrook molecular cloning book as mentioned earlier
Result
part | Concentration(ng/µl) |
BBa_K823016 | 2650.8 |
BBa_K1509003 | 4080.3 |
This has high RNA contamination
Gel Electrophoresis is done which shows high contamination of RNA
2/09/15
Inoculation of both BBa_K823016 and BBa_K1509003
Plasmid isolation using QIAGEN Miniprep spin column
Protocol: QIAGEN Miniprep spin column protocol is used
Result:
part | Concentration(ng/µl) |
BBa_K823016 | 275.2 |
BBa_K1509003 | 87.3 |
Gel Electrophoresis is done to check isolated plasmid
8/09/15
Inoculation of BBa_K1509003 in 5ml LB and 5µl cam
9/09/15
Gel Electrophoresis (1%)
Chemicals required: 0.5gm agarose, 50ml TAE, 5µl EtBr
Inoculation of both BBa_K823016 and BBa_K1509003 in 5ml LB each
10/09/15
Plasmid isolation using QIAGEN Miniprep spin column
Protocol: QIAGEN Miniprep spin column protocol is used
Result:
part | Concentration(ng/µl) |
BBa_K823016 | 735 |
BBa_K1509003 | 234 |
Single cut digestion
BBa_K823016: 1µl
BBa_K1509003:3µl
BBa_K823016 (µl) | BBa_K1509003 (µl) | |
DNA | 1 | 3 |
ECoR1 buffer | 1 | 1 |
ECoR1 enzyme | 0.5 | 0.5 |
dH2O | 7.5 | 5.5 |
Total | 10 | 10 |
Gel Electrophoresis: Fig1
12/09/15
Fig2
Confirmation of plasmid isolated
12/09/15
Digestion for 3A assembly
pSB1K3 which is a Kan backbone used for 3A assembly
BBa_K823016 (µl) | BBa_K1509003 (µl) | ||
1 | NEB buffer 2 | 5 | 5 |
2 | BSA | 0.5 | 0.5 |
3 | EcoR1-HF | 0.5 | 0.5 |
4 | Spel | 0.5 | 0.5 |
5 | dH20 | 18.5 | 18.5 |
6 | Total | 20 | 20 |
Protocol: standard iGEM 3A assembly protocol is used
13/09/15
Ligation using 3A assembly of biobricks
Protocol: standard iGEM 3A assembly protocol is used
15/09/15
Transformation of ligated parts
Protocol: IGEM transformation protocol is used
Result: No colony is observed on both KAN media plates
Comment: most probably failure of digestion
16/09/15
Inoculation of both BBa_K823016 and BBa_K1509003
Plasmid isolation using QIAGEN Miniprep spin column
Protocol: QIAGEN Miniprep spin column protocol is used
Result:
part | Concentration(ng/µl) |
BBa_K823016 | 102.3 |
BBa_K1509003 | 65.5 |