Team:IIT Kharagpur/Notebook
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PROTOCOL
WEEK ONE - ( 20th July - 22th July )
20th july, Wednesday
DH5 alpha competent cells were prepared for transformation experiments.
21st July, Thursday
Transformation of the prepared competent cells was carried out.
The plasmids used for transformation were YFP (BBa_K592101), HIV cleavage site (BBa_I712015), and the protease construct (BBa_K743013)
22nd July, Friday
Replication plating for above transformed parts was done. This including the three sets of transformed cells containing the plasmids YFP (BBa_K592101), the HIV cleavage site (BBa_I712015), and the protease construct (BBa_K743013).
20th july, Wednesday
DH5 alpha competent cells were prepared for transformation experiments.
21st July, Thursday
Transformation of the prepared competent cells was carried out. The plasmids used for transformation were YFP (BBa_K592101), HIV cleavage site (BBa_I712015), and the protease construct (BBa_K743013)
22nd July, Friday
Replication plating for above transformed parts was done. This including the three sets of transformed cells containing the plasmids YFP (BBa_K592101), the HIV cleavage site (BBa_I712015), and the protease construct (BBa_K743013).
WEEK TWO - ( 25th July - 30th July )
25th july, Monday
1 litre of LB media was prepared and duly autoclaved.
5 plates each of the antibiotics chloramphenicol, ampicillin and ampicillin+Kanamycin were prepared.
26th July, Tuesday
20ml Cultures were prepared for the plasmid extraction of each of YFP (BBa_K592101), the HIV cleavage site (BBa_I712015) and the protease construct (BBa_K743013).
27th July, Wednesday
Plasmid extraction was done for YFP (BBa_K592101), the HIV cleavage site (BBa_I712015), and the protease construct (BBa_K743013) using mini-prep plasmid DNA extraction kit.
28th July, Thursday
DNA quantification was done using Nano Drop.
YFP: 250 ng/ul
Protease: 170 ng/ul
Cleavage Site: 120 ng/ul
1% agarose gel was prepared for screening of the plasmids extracted by the mini prep kit.
The FRET construct was kept for overnight digestion at the restriction sites E and P.
Digestions with E and P for YFP, Protease and Cleavage Site
YFP Protease Cleavage Site Component Volume(ul) Volume(ul) Volume(ul) DNA 2 3 3.5 2.1 Buffer 2 2 2 ECoRI 0.5 0.5 0.5 Psti 0.5 0.5 - Nf H2O 15 14 14
25th july, Monday
1 litre of LB media was prepared and duly autoclaved. 5 plates each of the antibiotics chloramphenicol, ampicillin and ampicillin+Kanamycin were prepared.
26th July, Tuesday
20ml Cultures were prepared for the plasmid extraction of each of YFP (BBa_K592101), the HIV cleavage site (BBa_I712015) and the protease construct (BBa_K743013).
27th July, Wednesday
Plasmid extraction was done for YFP (BBa_K592101), the HIV cleavage site (BBa_I712015), and the protease construct (BBa_K743013) using mini-prep plasmid DNA extraction kit.
28th July, Thursday
DNA quantification was done using Nano Drop.
YFP: 250 ng/ul
Protease: 170 ng/ul
Cleavage Site: 120 ng/ul
1% agarose gel was prepared for screening of the plasmids extracted by the mini prep kit.
The FRET construct was kept for overnight digestion at the restriction sites E and P.
Digestions with E and P for YFP, Protease and Cleavage Site
| YFP | Protease | Cleavage Site | |
|---|---|---|---|
| Component | Volume(ul) | Volume(ul) | Volume(ul) |
| DNA | 2 | 3 | 3.5 |
| 2.1 Buffer | 2 | 2 | 2 |
| ECoRI | 0.5 | 0.5 | 0.5 |
| Psti | 0.5 | 0.5 | - |
| Nf H2O | 15 | 14 | 14 |
WEEK THREE - ( 1st August - 7th August )
1st August, Monday
3ml cultures made for the preparation of competent cells of DH5 alpha cells.
20ml culture prepared for the construct BBa_K844004. (MaSP without ATG)
2nd August, Tuesday
11 vials of DH5 alpha competent cells were prepared.
Plasmid extraction done for the construct BBa_K844004 (MaSP without ATG).
3rd August, Wednesday
As a part of the iGEM 2016 interlab study, transformations were done for the following:
Test device1
Test device2
Test device3
Positive control
Negative control
Also, transformed cells were prepared with the part BBa_K844008 (MaSP with ATG)
4th August, Thursday
Colonies were seen for all six of the transformed plates. The transformation procedure was hence successful.
7th August, Sunday
20ml Culture was prepared for the screening of BBa_K844008 (MaSP with ATG).
Also, 20ml cultures were prepared for each of test device 1, test device 2, and test device 3 for the interlab study.
1st August, Monday
3ml cultures made for the preparation of competent cells of DH5 alpha cells. 20ml culture prepared for the construct BBa_K844004. (MaSP without ATG)
2nd August, Tuesday
11 vials of DH5 alpha competent cells were prepared. Plasmid extraction done for the construct BBa_K844004 (MaSP without ATG).
3rd August, Wednesday
As a part of the iGEM 2016 interlab study, transformations were done for the following:
Test device1
Test device2
Test device3
Positive control
Negative control
Also, transformed cells were prepared with the part BBa_K844008 (MaSP with ATG)
4th August, Thursday
Colonies were seen for all six of the transformed plates. The transformation procedure was hence successful.
7th August, Sunday
20ml Culture was prepared for the screening of BBa_K844008 (MaSP with ATG). Also, 20ml cultures were prepared for each of test device 1, test device 2, and test device 3 for the interlab study.
WEEK FOUR - ( 8th August - 12th August )
8th August, Monday
PPlasmid extraction performed for the screening of the parts BBa_K844008 (MaSP with ATG); and test device 1, test device 2, test device 3 for the interlab study.
9th August, Tuesday
Gel Electrophoresis with 1% agarose gel was done for BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3 for screening.
The run was not successful due to gel over flow.
10th August, Wednesday
20ml Culture was prepared for screening of BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3.
11th August, Thursday
Plasmid extraction for the screening of BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3 was done.
12th August, Friday
Gel electrophoresis with 1% agarose gel was done for BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3.
8th August, Monday
PPlasmid extraction performed for the screening of the parts BBa_K844008 (MaSP with ATG); and test device 1, test device 2, test device 3 for the interlab study.
9th August, Tuesday
Gel Electrophoresis with 1% agarose gel was done for BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3 for screening. The run was not successful due to gel over flow.
10th August, Wednesday
20ml Culture was prepared for screening of BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3.
11th August, Thursday
Plasmid extraction for the screening of BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3 was done.
12th August, Friday
Gel electrophoresis with 1% agarose gel was done for BBa_K844008 (MaSP with ATG), test device 1, test device 2, test device 3.
WEEK FIVE - ( 15th August - 18th August )
16th August, Tuesday
Plasmid extraction of BBa_K844008 (MaSP with ATG), test device 1, test device 2, and test device 3 was done with mini-prep kit.
Digestion of above obtained plasmids with E And P was carried out.
17th August, Wednesday
1% agarose gel was prepared for digested plasmids
PCR of protease construct for HIV site primers was done
PCR parameters:
{95 degree 2 minutes
[95 degree 30 seconds
60 degrees 30 sec
68 degree 2 min] X35 cycles
68 degree 10 min}
PCR products run in 1% agarose gel.
Amplification not upto expectation.
Repetition of the experiment, with different conditions suggested.
Note: Too many non specific products were observed that is why the same was repeated next week.
18th August, Thursday
PCR of protease construct for HIV site primers was done.
PCR products were run in 1% agarose gel.
PCR Parameters:
{95 degree 2 minutes
[95 degree 20 seconds
62 degrees 30 sec
68 degree 2 min 15 sec] X35 cycles
68 degree 10 min}
Amplification not upto expectation.
Repetition of the experiment, with different conditions suggested.
16th August, Tuesday
Plasmid extraction of BBa_K844008 (MaSP with ATG), test device 1, test device 2, and test device 3 was done with mini-prep kit. Digestion of above obtained plasmids with E And P was carried out.
17th August, Wednesday
1% agarose gel was prepared for digested plasmids
PCR of protease construct for HIV site primers was done
PCR parameters:
{95 degree 2 minutes
[95 degree 30 seconds
60 degrees 30 sec
68 degree 2 min] X35 cycles
68 degree 10 min}
PCR products run in 1% agarose gel.
Amplification not upto expectation.
Repetition of the experiment, with different conditions suggested.
Note: Too many non specific products were observed that is why the same was repeated next week.
18th August, Thursday
PCR of protease construct for HIV site primers was done.
PCR products were run in 1% agarose gel.
PCR Parameters:
{95 degree 2 minutes
[95 degree 20 seconds
62 degrees 30 sec
68 degree 2 min 15 sec] X35 cycles
68 degree 10 min}
Amplification not upto expectation.
Repetition of the experiment, with different conditions suggested.
WEEK SIX - ( 22nd August - 26th August )
22th August, Monday
PCR for both fret and silk constructs at two conditions was done.
Condition 1
{95 degree 5 minutes
Taq DNA polymerase is added.
[95 degree 30 seconds
57.5 degrees 40 sec
68 degree 40 sec] X35 cycles
68 degree 10 min}
Condition 2
{95 degree 2 minutes
[95 degree 30 seconds
58 degrees 1 min
68 degree 2 min] X35 cycles
68 degree 10 min}
23rd August, Tuesday
PCR for FRET construct was done.
PCR Parameters:
{95 degree 2 minutes
[95 degree 30 seconds
57 degrees 40 sec
68 degree 2 min] X35 cycles
68 degree 10 min}
24th August, Wednesday
Gel run for FRET construct was performed.
25th August, Thursday
PCR for silk construct was done with the following parameters:
{95 degree 2 minutes
[95 degree 30 seconds
57 degrees 40 sec
68 degree 2 min] X35 cycles
68 degree 10 min}
26th August, Friday
PCR for FRET construct was done.
PCR Parameters:
{95 degree 2 minutes
[95 degree 30 seconds
58 degrees 1 min
68 degree 2 min] X35 cycles
68 degree 10 min}
22th August, Monday
PCR for both fret and silk constructs at two conditions was done.
Condition 1
{95 degree 5 minutes
Taq DNA polymerase is added.
[95 degree 30 seconds
57.5 degrees 40 sec
68 degree 40 sec] X35 cycles
68 degree 10 min}
Condition 2
{95 degree 2 minutes
[95 degree 30 seconds
58 degrees 1 min
68 degree 2 min] X35 cycles
68 degree 10 min}
23rd August, Tuesday
PCR for FRET construct was done.
PCR Parameters:
{95 degree 2 minutes
[95 degree 30 seconds
57 degrees 40 sec
68 degree 2 min] X35 cycles
68 degree 10 min}
24th August, Wednesday
Gel run for FRET construct was performed.
25th August, Thursday
PCR for silk construct was done with the following parameters:
{95 degree 2 minutes
[95 degree 30 seconds
57 degrees 40 sec
68 degree 2 min] X35 cycles
68 degree 10 min}
26th August, Friday
PCR for FRET construct was done.
PCR Parameters:
{95 degree 2 minutes
[95 degree 30 seconds
58 degrees 1 min
68 degree 2 min] X35 cycles
68 degree 10 min}
WEEK SEVEN - ( 29th August - 2nd September )
29th August, Monday
Nano drop analysis for pSB1K3( Kanamycin backbone) was done.
Result:Obtained concentration was 192.6mg/ul.
31st August, Wednesday
Digestion for Kanamycin backbone and FRET PCR with E And P
K backbone FRET PCR Component Volume(ul) Volume(ul) DNA 6 2.3 2.1 Buffer 2 2 ECoRI 1 1 Psti 1 1 Nf H2O 10 13.7
1st September, Thursday
Ligation for Kanamycin backbone and FRET PCR with E and P was done
Vector:Insert=1:3
2nd September, Friday
Transformation for Kanamycin backbone and FRET PCR with E and P failed.
Note: Reason was determined to be inadequate amount of the PCR product
29th August, Monday
Nano drop analysis for pSB1K3( Kanamycin backbone) was done. Result:Obtained concentration was 192.6mg/ul.
31st August, Wednesday
Digestion for Kanamycin backbone and FRET PCR with E And P
| K backbone | FRET PCR | |
|---|---|---|
| Component | Volume(ul) | Volume(ul) |
| DNA | 6 | 2.3 |
| 2.1 Buffer | 2 | 2 |
| ECoRI | 1 | 1 |
| Psti | 1 | 1 |
| Nf H2O | 10 | 13.7 |
1st September, Thursday
Ligation for Kanamycin backbone and FRET PCR with E and P was done Vector:Insert=1:3
2nd September, Friday
Transformation for Kanamycin backbone and FRET PCR with E and P failed.
Note: Reason was determined to be inadequate amount of the PCR product
WEEK EIGHT - ( 8th September - 10th September )
8th September, Thursday
2 ml culture of CFP and YFP was prepared.
PCR done for FRET construct and silk construct
Digestion with E and P of FRET PCR product was done.
FRET PCR Component Volume(ul) DNA 19 2.1 Buffer 2.5 ECoRI 1 PstI 1 NF H2O 1.5
9th September, Friday
PCR purification of digested FRET PCR product was done.
Plasmid isolation of YFP and CFP was performed.
YFP: 250 ng/ul
CFP: 218 ng/ul
Glycerol stock for CFP and YFP was prepared.
Restriction digestion(overnight) of YFP and cleavage site was done.
YFP Component Volume(ul) DNA 5 ECoR buffer 2 ECoRI 1 NF H2O 12
Cleavage Site Component Volume(ul) DNA 10 3.1 Buffer 2 ECoRI 1 Xbal 1 NF H2O 6
10th September, Saturday
PCR purification of YFP digested with E was done.
Digestion of purified YFP was done with S for 4 hrs at 37 degrees.
YFP Component Volume(ul) DNA 20 Cutsmart Buffer 2.5 Spel 1 NF H2O 1.5
8th September, Thursday
2 ml culture of CFP and YFP was prepared.
PCR done for FRET construct and silk construct
Digestion with E and P of FRET PCR product was done.
| FRET PCR | |
|---|---|
| Component | Volume(ul) |
| DNA | 19 |
| 2.1 Buffer | 2.5 |
| ECoRI | 1 |
| PstI | 1 |
| NF H2O | 1.5 |
9th September, Friday
PCR purification of digested FRET PCR product was done.
Plasmid isolation of YFP and CFP was performed.
YFP: 250 ng/ul
CFP: 218 ng/ul
Glycerol stock for CFP and YFP was prepared.
Restriction digestion(overnight) of YFP and cleavage site was done.
| YFP | |
|---|---|
| Component | Volume(ul) |
| DNA | 5 |
| ECoR buffer | 2 |
| ECoRI | 1 |
| NF H2O | 12 |
| Cleavage Site | |
|---|---|
| Component | Volume(ul) |
| DNA | 10 |
| 3.1 Buffer | 2 |
| ECoRI | 1 |
| Xbal | 1 |
| NF H2O | 6 |
10th September, Saturday
PCR purification of YFP digested with E was done. Digestion of purified YFP was done with S for 4 hrs at 37 degrees.
| YFP | |
|---|---|
| Component | Volume(ul) |
| DNA | 20 |
| Cutsmart Buffer | 2.5 |
| Spel | 1 |
| NF H2O | 1.5 |
WEEK NINE - ( 12th September - 13th September )
12th September, Monday
22 ml cultures of CFP, YFP, HIV site, MASP with ATG and MASP without ATG was prepared.
13th September, Tuesday
Glycerol stock and plasmid extraction of CFP, YFP, HIV site, MASP with ATG and MASP without ATG was performed.
CFP:437.5 ng/ul
YFP: 286.7 ng/ul
HIV Cleavage Site: 357.1 ng/ul
MaSP with ATG: 268.2 ng/ul
MaSP w/o ATG: 123ng/ul
12th September, Monday
22 ml cultures of CFP, YFP, HIV site, MASP with ATG and MASP without ATG was prepared.
13th September, Tuesday
Glycerol stock and plasmid extraction of CFP, YFP, HIV site, MASP with ATG and MASP without ATG was performed.
CFP:437.5 ng/ul
YFP: 286.7 ng/ul
HIV Cleavage Site: 357.1 ng/ul
MaSP with ATG: 268.2 ng/ul
MaSP w/o ATG: 123ng/ul
WEEK TEN - ( 19th September - 24th September )
19th September, Monday
PCR for FRET (2 sets)
Digestion with E and X for HIV site and E for CFP was successfully done.
CFP Component Volume(ul) DNA 20 ECoR buffer 2 ECoRI 1 NF H2O 14.7
Cleavage Site Component Volume(ul) DNA 2.8 3.1 Buffer 2 ECoRI 1 Xbal 1 NF H2O 13.2
20th September, Tuesday
PCR purification of CFP digested with E was done.
Digestion with S of purified product was done for 6 hours at 37 degrees.
CFP Component Volume(ul) DNA 20 Cutsmart Buffer 2.5 Spel 1 NF H2O 1.5
Gel extraction of digested CFP and HIV site was successfully performed.
The gel pieces stored at 4 degrees
21st September, Wednesday
Gel extraction of stored gel pieces
Amount of extracted DNA too low to proceed to ligation step
22nd September, Thursday
S digestion of CFP and X digestion of HIV
23rd September, Friday
PCR purification of above digested followed by E digestion of both for 6 hrs at 37 degrees
Gel extraction after completion of digestion followed by ligation of E and X digested HIV site and E and S digested CFP at 16 degrees overnight
24th September, Saturday
Transformed DH5 alpha cells with ligation mix and plating on A+K plates
19th September, Monday
PCR for FRET (2 sets)
Digestion with E and X for HIV site and E for CFP was successfully done.
| CFP | |
|---|---|
| Component | Volume(ul) |
| DNA | 20 |
| ECoR buffer | 2 |
| ECoRI | 1 |
| NF H2O | 14.7 |
| Cleavage Site | |
|---|---|
| Component | Volume(ul) |
| DNA | 2.8 |
| 3.1 Buffer | 2 |
| ECoRI | 1 |
| Xbal | 1 |
| NF H2O | 13.2 |
20th September, Tuesday
PCR purification of CFP digested with E was done. Digestion with S of purified product was done for 6 hours at 37 degrees.
| CFP | |
|---|---|
| Component | Volume(ul) |
| DNA | 20 |
| Cutsmart Buffer | 2.5 |
| Spel | 1 |
| NF H2O | 1.5 |
Gel extraction of digested CFP and HIV site was successfully performed. The gel pieces stored at 4 degrees
21st September, Wednesday
Gel extraction of stored gel pieces Amount of extracted DNA too low to proceed to ligation step
22nd September, Thursday
S digestion of CFP and X digestion of HIV
23rd September, Friday
PCR purification of above digested followed by E digestion of both for 6 hrs at 37 degrees Gel extraction after completion of digestion followed by ligation of E and X digested HIV site and E and S digested CFP at 16 degrees overnight
24th September, Saturday
Transformed DH5 alpha cells with ligation mix and plating on A+K plates
WEEK ELEVEN - ( 25th September - 2nd October )
25th September, Sunday
Colonies observed on incubated plates
26th September, Mondayy
18 colonies from the 1:3 plate were put into 1 ml media overnight for screening
14 colonies from the 1:5 plate were put into 1 ml media overnight for screening
Digestion of YFP with X and P
Digestion of HIV site with S and P
27th September, Tuesday
PCR for FRET (2 sets)
Gel extraction for PCR products and digestion with P
Digestion of K backbone with P
Screening for 18 colonies from the 1:3 plate and 14 colonies from the 1:5 plate
Colonies 1, 4, 7, 10 from 1:3 plate showed a shift
Colonies 1, 5, 11 from 1:5 plate showed a shift
Ligation of YFP digested with X and P with HIV site digested with S and P
28th September, Wednesday
PCR purification of P digested K backbone and FRET PCR followed by E digestion
Gel Extraction of the digested products followed by ligation
Colonies observed of ligated YFP and HIV site observed
29th September, Thursday
25 colonies from the ligated YFP and HIV site plate picked for screening
X and P digestion for CFP+ HIV
30th September, Friday
Screening of YFP + HIV
Gel run X and P digested CFP + HIV
20 ml cultures for plasmid extraction of K-backbone, CFP+HIV and HIV Site, YFP+HIV (colony 7) and YFP+HIV (colony 13)
1st October, Saturday
Plasmid extractions for the above cultures
Digestion with X and P for YFP+HIV (colony 7) and CFP+HIV
Gel run for the digested YFP+HIV (colony 7) and CFP+HIV
Digestion with X of YFP+HIV
Digestion with S of CFP
2nd October, Sunday
PCR purification of the digested products
Digestion with E of S digested CFP and X digested YFP+HIV
Gel extraction of the digested product
Ligation of E and S digested CFP and E and X digested YFP+HIV
25th September, Sunday
Colonies observed on incubated plates
26th September, Mondayy
18 colonies from the 1:3 plate were put into 1 ml media overnight for screening
14 colonies from the 1:5 plate were put into 1 ml media overnight for screening
Digestion of YFP with X and P
Digestion of HIV site with S and P
27th September, Tuesday
PCR for FRET (2 sets)
Gel extraction for PCR products and digestion with P
Digestion of K backbone with P
Screening for 18 colonies from the 1:3 plate and 14 colonies from the 1:5 plate
Colonies 1, 4, 7, 10 from 1:3 plate showed a shift
Colonies 1, 5, 11 from 1:5 plate showed a shift
Ligation of YFP digested with X and P with HIV site digested with S and P
28th September, Wednesday
PCR purification of P digested K backbone and FRET PCR followed by E digestion Gel Extraction of the digested products followed by ligation Colonies observed of ligated YFP and HIV site observed
29th September, Thursday
25 colonies from the ligated YFP and HIV site plate picked for screening X and P digestion for CFP+ HIV
30th September, Friday
Screening of YFP + HIV
Gel run X and P digested CFP + HIV
20 ml cultures for plasmid extraction of K-backbone, CFP+HIV and HIV Site, YFP+HIV (colony 7) and YFP+HIV (colony 13)
1st October, Saturday
Plasmid extractions for the above cultures
Digestion with X and P for YFP+HIV (colony 7) and CFP+HIV
Gel run for the digested YFP+HIV (colony 7) and CFP+HIV
Digestion with X of YFP+HIV
Digestion with S of CFP
2nd October, Sunday
PCR purification of the digested products
Digestion with E of S digested CFP and X digested YFP+HIV
Gel extraction of the digested product
Ligation of E and S digested CFP and E and X digested YFP+HIV
