E. coli were grown in LB+ Amp.100 mg/ml over night.
Tuesday, December 16, 2014
Wednesday, December 10, 2014
Cosmid purification from pDRTn1,2,3,4,,5,6,7,8 and 9
Nov.3\2014
Using the fallowing protocol http://www.epibio.com/docs/default-source/protocols/fosmidmax-dna-purification-kit.pdf?sfvrsn=8
Using the fallowing protocol http://www.epibio.com/docs/default-source/protocols/fosmidmax-dna-purification-kit.pdf?sfvrsn=8
Thursday, December 4, 2014
Transformation of Chemically competent E. coli
Date Dec./4/2014
The fallowing protocol where used from invitrogen
One Shot® Mach1™-T1R Chemically Competent E. coli
Transforming Competent Cells
Perform the following before starting the transformation procedure:
• Equilibrate a water bath to 42°C.
• Warm the vial of S.O.C. Medium (supplied with the kit) to room temperature.
• Spread X-Gal onto LB agar plates containing antibiotic, if desired.
• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100 μg/ml ampicillin. Note: For optimal growth of Mach1™-T1R E. coli cells, it is essential that selective plates are prewarmed to 37°C prior to spreading.
Transformation Procedure
We recommend including the pUC19 control plasmid DNA supplied with the kit in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.
1. Thaw, on ice, one vial of One Shot® Mach1™-T1R Chemically Competent E. coli for each transformation.
2. Add 1 to 5 μl of the DNA (10 pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. If you are transforming the pUC19 control, add 1 μl (10 pg) into a separate vial of One Shot® cells and mix gently.
3. Incubate the vial(s) on ice for 30 minutes.
4. Heat-shock the cells for 30 seconds at 42°C without shaking.
5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
6. Add 250 μl of room temperature S.O.C. Medium to each vial.
7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.
8. Spread 25-100 μl of the transformation mix on a prewarmed selective plate. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.
The fallowing protocol where used from invitrogen
One Shot® Mach1™-T1R Chemically Competent E. coli
Transforming Competent Cells
Perform the following before starting the transformation procedure:
• Equilibrate a water bath to 42°C.
• Warm the vial of S.O.C. Medium (supplied with the kit) to room temperature.
• Spread X-Gal onto LB agar plates containing antibiotic, if desired.
• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100 μg/ml ampicillin. Note: For optimal growth of Mach1™-T1R E. coli cells, it is essential that selective plates are prewarmed to 37°C prior to spreading.
Transformation Procedure
We recommend including the pUC19 control plasmid DNA supplied with the kit in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.
1. Thaw, on ice, one vial of One Shot® Mach1™-T1R Chemically Competent E. coli for each transformation.
2. Add 1 to 5 μl of the DNA (10 pg to 100 ng) into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down. If you are transforming the pUC19 control, add 1 μl (10 pg) into a separate vial of One Shot® cells and mix gently.
3. Incubate the vial(s) on ice for 30 minutes.
4. Heat-shock the cells for 30 seconds at 42°C without shaking.
5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
6. Add 250 μl of room temperature S.O.C. Medium to each vial.
7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.
8. Spread 25-100 μl of the transformation mix on a prewarmed selective plate. Store the remaining transformation mix at +4°C. Additional cells may be plated out the next day, if desired.
- E. coli strain DH 5 alfa with no plasmid and sensitive to Amp. inoculated on LB+Amp. used as a control.
- One Shot® Mach1™-T1R Chemically Competent E. coli was inoculated on LB agar only to test its viability.
Sunday, November 23, 2014
Dictyostelium Predation Assay for transposon mutagensis of pDR2 Part(2)
Date: 10/1/2014
The protocol in the link
Plate name : pDR 2/Kana. P1 & 4 , transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
Plaques in Kleb. plate=1 plaques in dilution 1/1000 (-3)
The result after 4 days :
Controls:
pDR2plague :( 0, -1, -2 )
cosmid control.plague(0,-1,-2,-3)
Clones shows sensitivity to Dictyostelium predation:
plague (0,-1,-2,-3)
The protocol in the link
Plate name : pDR 2/Kana. P1 & 4 , transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
Plaques in Kleb. plate=1 plaques in dilution 1/1000 (-3)
Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
=1.0 X10^3
2 μl of 8 Dicty. dilutions that contain(2.0 X 10^2, 2.0 X 10^1, 2 , 0.2,....)Dicty. cells were used on sm2 agar in four replicate for each dilution.=1.0 X10^3
The result after 4 days :
Controls:
pDR2plague :( 0, -1, -2 )
cosmid control.plague(0,-1,-2,-3)
Clones shows sensitivity to Dictyostelium predation:
plague (0,-1,-2,-3)
Dictyostelium Predation Assay for transposon mutagensis of pDR1 Part(4)
Date: Nov./10/2014
The protocol in the link
Plate name :collection of 3 paltes(contain 10 +20+10 colonies), pDR1/kana. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
The protocol in the link
Plate name :collection of 3 paltes(contain 10 +20+10 colonies), pDR1/kana. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
Plaques in Kleb. plate=1 plaques in dilution 1/1000 (-3)
Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
=1.0 X10^3
2 μl of 8 Dicty. dilutions that contain(2.0 X 10^2, 2.0 X 10^1, 2 , 0.2,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
9-2A8 named: pDR1tn9
=1.0 X10^3
2 μl of 8 Dicty. dilutions that contain(2.0 X 10^2, 2.0 X 10^1, 2 , 0.2,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
- pDR1plague :(0)
- cosmid control.plague(0,-1,-2)
- Clones shows sensitivity to Dictyostelium predation:
9-2A8 named: pDR1tn9
Monday, November 10, 2014
Dictyostelium Predation Assay for transposon mutagensis of pDR2 Part(1)
Date: Nov./1/2014
The protocol in the link
Plate name : pDR 2/Tetra. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
The protocol in the link
Plate name : pDR 2/Tetra. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
Plaques in Kleb. plate=35 plaques in dilution 1/1000 (-3)
Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
35 X10^3/10 = 3.5 X10^3
2 μl of 8 Dicty. dilutions that contain(7.0 X 10^3, 7.0 X 10^2, 7.0 X 10 ,7.0 , 0.7,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
A7 named: pDR2 tn 1
A8 named: pDR2 tn 2
A12 named: pDR2 tn 3
B3 named: pDR2 tn 41
C4 named: pDR2 tn 5
D3 named: pDR2 tn 6
D11 named: pDR2 tn 7
E2 named: pDR2 tn 8
E5 named: pDR2 tn 9
E6 named: pDR2 tn 10
E8 named: pDR 2 tn 11
plague (0,-1,-2,-3,-4)
E4 named: pDR2tn12
35 X10^3/10 = 3.5 X10^3
2 μl of 8 Dicty. dilutions that contain(7.0 X 10^3, 7.0 X 10^2, 7.0 X 10 ,7.0 , 0.7,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
- pDR2plague :( 0, -1, -2 )
- cosmid control.plague(0,-1,-2,-3,-4)
- Clones shows sensitivity to Dictyostelium predation:
A7 named: pDR2 tn 1
A8 named: pDR2 tn 2
A12 named: pDR2 tn 3
B3 named: pDR2 tn 41
C4 named: pDR2 tn 5
D3 named: pDR2 tn 6
D11 named: pDR2 tn 7
E2 named: pDR2 tn 8
E5 named: pDR2 tn 9
E6 named: pDR2 tn 10
E8 named: pDR 2 tn 11
plague (0,-1,-2,-3,-4)
E4 named: pDR2tn12
Thursday, October 30, 2014
Dictyostelium Predation Assay for transposon mutagensis of pDR1 Part(3)
Date: Oct./28/2014
The protocol in the link
Plate name : individual colonies(contain 10 colonies), pDR1/kana. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
The protocol in the link
Plate name : individual colonies(contain 10 colonies), pDR1/kana. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
Plaques in Kleb. plate=32 plaques in dilution 1/1000 (-3)
Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
32 X10^3/10 = 3.2 X10^3
2 μl of 8 Dicty. dilutions that contain(6.4 X 10^3, 6.4 X 10^2, 6.4 X 10 ,6.4 , 0.64,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
8-A5 named: pDR1tn8
32 X10^3/10 = 3.2 X10^3
2 μl of 8 Dicty. dilutions that contain(6.4 X 10^3, 6.4 X 10^2, 6.4 X 10 ,6.4 , 0.64,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
- pDR1plague :(0,-1)
- cosmid control.plague(0,-1,-2,-3,-4)
- Clones shows sensitivity to Dictyostelium predation:
8-A5 named: pDR1tn8
Dictyostelium Predation Assay for transposon mutagensis of pDR1 Part(2)
Date: Oct./25/2014
The protocol in the link
Plate 4, pDR1/kana. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
The protocol in the link
Plate 4, pDR1/kana. transposon mutagensis.
The result: after 4 days incubation at room temp. in a dark place
Plaques in Kleb. plate=23 plaques in dilution 1/1000 (-3)
Plaque forming unit pfu =# plaque X dilution factor/volume of inoculum
23 X10^3/10 = 2.3 X10^3
2 μl of 8 Dicty. dilutions that contain(4.6 X 10^3, 4.6 X 10^2,4.6 X 10 ,4.6 , 0.46 ,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
6-A7 named: pDR1tn6 - small palques-
plague(0,-1,-2,-3,-4)
7-A6 named: pDR1tn6
The result after 5 days incubation look like same
23 X10^3/10 = 2.3 X10^3
2 μl of 8 Dicty. dilutions that contain(4.6 X 10^3, 4.6 X 10^2,4.6 X 10 ,4.6 , 0.46 ,....) Dicty. cells were used on sm2 agar in four replicate for each dilution.
The result after 4 days :
Controls:
- pDR1plague :(0,-1,-2)
- cosmid control.plague(0,-1,-2,-3,-4)
- Clones shows sensitivity to Dictyostelium predation:
6-A7 named: pDR1tn6 - small palques-
plague(0,-1,-2,-3,-4)
7-A6 named: pDR1tn6
The result after 5 days incubation look like same
Thursday, October 23, 2014
Plaques measurement by Dicty. predation assay
The result
In Kleb. PFU= (average # of plaques
x d.f)/volume of culture
=6 X 105 /10 = 6.0
X 104
3 different dilution of Dicty. in sterile distal water( 0, 1/100,
1/10000) were used on different clones
and strains of bacteria. Each clone or strain was inoculated on sm2 agar
2 μl that contain dose of (1.6 X10^3, 1.6 X 10^2 , 1.6 X 10 , 1.6 , 0.16 ) of each Dicty. dilution titration containing(3.0 X 105 ,3.0 X103 and 3.0 X 10) was dropped
on sm2 agar that have been inoculated previously with the specific bacterial
clone, and plate were kept at room temperature in a dark place.
After
3 days incubation, the diameter of plaque in mm
Bacterial strain
|
0 Dicty. dilution
|
1:100 dilution
|
1:10000 dilution
|
|
DH5 alfa E.coli
|
11
|
5
|
-
|
|
EPI 100 E.coli
|
10.5
|
5
|
-
|
|
Klebsiella.
|
13
|
5
|
-
|
|
pDR Cosmid control
|
11
|
5
|
-
|
|
pDR1
|
3.5
|
3
|
-
|
|
pDR2
|
3
|
-
|
-
|
|
pDR4
|
5
|
-
|
-
|
|
pDR5
|
3
|
-
|
-
|
|
pDR6
|
-
|
-
|
-
|
|
Stenotrophomonas maltophilia BAA2423(K279a)
|
5.5
|
-
|
-
|
|
S.m BAA84(D457)
|
5
|
-
|
-
|
|
S.m M
|
5
|
-
|
-
|
|
S.m N
|
-
|
-
|
-
|
|
S.m H
|
4
|
-
|
-
|
|
After
4 days incubation, the diameter of plaque in mm
Bacterial strain
|
0 Dicty. dilution
|
1:100 dilution
|
1:10000 dilution
|
|
DH5 alfa E.coli
|
18
|
12
|
-
|
|
EPI 100 E.coli
|
19
|
8
|
8
|
|
Klebsiella.
|
30
|
16
|
10
|
|
pDR Cosmid control
|
18
|
12
|
9
|
|
pDR1
|
14
|
9
|
-
|
|
pDR2
|
5
|
-
|
-
|
|
pDR4
|
8
|
-
|
-
|
|
pDR5
|
7
|
-
|
-
|
|
pDR6
|
9
|
-
|
-
|
|
Stenotrophomonas maltophilia BAA2423(K279a)
|
9
|
-
|
-
|
|
S.m BAA84(D457)
|
7
|
-
|
-
|
|
S.m M
|
7
|
-
|
-
|
|
S.m N
|
3
|
-
|
-
|
|
S.m H
|
7
|
-
|
-
|
|
After
5 days incubation, the diameter of plaque in mm
Bacterial strain
|
0 Dicty. dilution
|
1:100 dilution
|
1:10000 dilution
|
|
DH5 alfa E.coli
|
18
|
17
|
8
|
|
EPI 100 E.coli
|
30
|
10
|
13
|
|
Klebsiella.
|
40
|
25
|
17
|
|
pDR Cosmid
control
|
25
|
17
|
11
|
|
pDR1
|
14
|
10
|
5
|
|
pDR2
|
5
|
-
|
-
|
|
pDR4
|
8
|
-
|
-
|
|
pDR5
|
9
|
-
|
-
|
|
pDR6
|
10
|
-
|
-
|
|
Stenotrophomonas
maltophilia BAA2423(K279a)
|
18
|
8
|
-
|
|
S.m BAA84(D457)
|
7
|
-
|
-
|
|
S.m
M
|
9
|
-
|
-
|
|
S.m
N
|
3
|
-
|
-
|
|
S.m H
|
9
|
-
|
-
|
|
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