Team:Heidelberg/Templates/DelH week9

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Revision as of 20:55, 1 October 2013

24-06 - 30-06-13

Characterization of DelH plasmid 19-06

SDS Page

Using NuPAGE Novex 10% Bis-Tris precast gel from Invitrogen with MOPS running buffer

3NuPAGE Novex 3-8% Tris-Acetate precast gel would have been more suitable considering expected 600 kDa DelH, but needed Tris-Acetate SDS buffer was not available.

Resuspended pellets in 100 µl SDS buffer (from Linda)
Boiled for 10 min @98°C
Ran for 90 min, 180 V
Stained for 40 min in Coomassie Buffer (50 ml acetic acid 100% + 225 ml ddH₂O, 1.5 g Coomassie in 225 ml methanol, mix both)
Washed in ddH₂O multiple times

Result

Fig.9.1 SDS PAGE
L1: Ladder L2: 6 µl of Konrad’s E. coli L3: 6 µl DelH colony 4 L4: 6 µl DelH colony 6 L5: 9 µl of Konrad’s E. coli L6: 9 µl DelH colony 4 L7: 9 µl DelH colony 6 L8: 15 µl of Konrad’s E. coli L9: 15 µl DelH colony 4 L10: 15 µl DelH colony 6 L11: empty L12: Ladder

None clear band at ~600 kDa visible. Maybe in highest amounts, but not reliable.

=> Probably no DelH expression in analyzed colonies.

Mini Prep

Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9)
Incubation ON at 37°C
5 ml ON cultures were mini prepared and resuspended in 50 µl

Test Restriction Digest

5 µl of the mini prep were test digested

Reagent	Volume [µl]
Miniprep DNA	5
CutSmart buffer	2
EcoRI-HF, PacI, KpnI-HF	0.5 each
ddH₂O	10.75

Incubation at 37°C for 1 h

Result

Expected band pattern: 5 kp, 7.3 kp, 13 kp

Fig.9.2 Gel of amplified fragments (loaded 20 µL)
l1: 2 log ladder, l2:test digested DelH fragment
l2: no visible band

No clear bands visible.

=> DNA yield from mini prep was probably too low.

DNA Concentration

Colony	DNA concentration [ng/µl]
1	124.5
4	3.9
6	1.6
9	10.2

DNA measurement confirmed suspicion from test digest.

=>Thus, mini prep and test digest have to be repeated

Repetion of Mini Prep

Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9) or from remaining ON culture (colony 1)
Incubation ON at 37°C
Colony 1 did not grow (also not on LB Amp plate), obviously lost plasmid
Mini preped remaining 3, eluted in 35 µl ddH₂O

DNA Concentration

Colony	DNA concentration [ng/µl]
4	38.5
6	42.0
9	49.0

Test Restriction Digest

Digested entire mini prep (using remaining from first mini for colony 1) for 2 h at37°C

Reagent	Volume [µl]
Mini prep	42
CutSmart	5
EcoRI-HF, PacI, KpnI-HF	1 each

Result

Expected bands: BB (7.3 Kb), DelH F2 (8 Kb), F1a + F1b (10 Kb)

Fig.9.3 miniprep resitriction digested (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1a
l2: DelH-F1a shows specific band = cut out

Unexpected bands at 3 Kb, but not desired ones.

=> Colonies do not contain desired plasmid and entire ligation has to be repeated.

Amplification of DelH F1b

PCR Conditions F1b.W9.A

Reagent	DelH F1b
Template	1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM	0.5 µl DelH_EcoRI_fw 100 µM
Primer rev 10 µM	0.5 µl DelH_f1_SalI_rev 100 µM
Phusion Ready Mix	25 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time
1	98	30 s
35	98	15 s
	68	5 s
	72	3:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.4 gel of amplified DelH 1b - fragment (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1b
l2: DelH-F1b shows no band

No correct band visible.

=> Why could we not reproduce the previous amplification?

PCR Conditions F1b.W9.B

Reagent	DelH F1b
Template	1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM	0.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM	0.5 µl DelH_f1_SalI_rev 10 µM
Phusion Ready Mix	25 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time
1	98	30 s
30	98	15 s
	68	15 s
	72	3:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.5 gel of amplified DelH-1b-fragment (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1b
l2: DelH-F1b shows unexpected band at 2.3 Kb

Again, expected band not there.

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.

PCR Conditions F1b.W9.C

Reagent	DelH F1b
Template	1 µl DelH F1b Fragment
Primer fw 10 µM	0.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM	0.5 µl DelH_f1_SalI_rev 10 µM
Phusion Flash Ready Mix	10 µl
ddH₂O	7 µl

Cycles	Temperature [°C]	Time
1	98	10 s
30	98	1 s
	68	5 s
	72	120
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 5 Kb

Fig.9.7 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 5 Kb = was cut out

Highly specific band at 5 Kb, as well as additional smaller ones.

=> Band was cut. Run gel with remaining sample for gel extraction.

Fig.9.8 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified

Fig.9.9 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified

PCR Conditions F1b.W9.D

Reagent	DelH F1b
Template	1 µl D. acidovorans stock 29-04-13 1:10
Primer fw 10 µM	0.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM	0.5 µl DelH_f1_SalI_rev 10 µM
Q5 Ready Mix	10 µl
ddH₂O	7 µl

Cycles	Temperature [°C]	Time
1	98	10 s
30	98	1 s
	68	5 s
	72	2:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: DelH F1b 5 Kb

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!

Amplification of DelH F2

PCR Conditions F2.W9.A

Reagent	DelH 2
Template	1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM	0.5 µl DelH_f2_SalI_fw 100 µM
Primer rev 10 µM	0.5 µl DelH_f2_KpnI_rev 100 µM
Phusion Ready Mix	25 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time
1	98	30 s
35	98	15 s
	66	5 s
	72	3:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.11 gel of amplified fragments (loaded 1 µL)
l1: 2log ladder, l2:F1b, l3: F2, l4: BB
shows band at 3 Kb

Unexpected bands at 3 Kb in lanes of DelH 2

=> Why could we not reproduce the previous amplification?

PCR Conditions F2.W9.B

Reagent	DelH 2
Template	1 µl D. acidovorans stock 29-04-13
Primer fw 10 µM	0.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM	0.5 µl DelH_f2_KpnI_rev 10 µM
Phusion Ready Mix	25 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time
1	98	30 s
12	98	15 s
	66 decrease by 0.5	15 s
	72	3:30 min
18	98	15 s
	66	15 s
	72	3:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.12 gel of amplified fragments (loaded 1 µL)
l1:2log, l2: F1b, l3: F2, l4: BB
F2 shows no specific band

Unexpected bands at 1.5 Kb, 2.2 Kb, 4 Kb and 6 Kb. Desired 8 Kb band is present but hardly visible.

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.

PCR Conditions F2.W9.C

Reagent	DelH 2
Template	1 µl D. acidovorans stock 29-04-13 1:10
Primer fw 10 µM	0.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM	0.5 µl DelH_f2_KpnI_rev 10 µM
Phusion Flash Ready Mix	10 µl
ddH₂O	7 µl

Cycles	Temperature [°C]	Time
1	98	10 s
12	98	1 s
	66 decrease by 0.5	5 s
	72	2:30 min
18	98	1 s
	66	5 s
	72	2:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l3: F2 shows specific band at 8 Kb and other ones

Fig.9.7 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 8 Kb = was cut out

F2 shows specific band at 8 Kb and additional ones.

=> Band was cut. Run gel with remaining sample for gel extraction.

Fig.9.8 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL)
l1:2log ladder, l2: F1a, l3:F2
l3: F2 shows specific band at 8 Kb and other ones

Fig.9.9 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL)
l1:2log ladder, l2: F1a, l3:F2
l3: F2 shows specific band at 8 Kb and other ones

PCR Conditions F2.W9.D

Reagent	DelH 2
Template	1 µl D. acidovorans stock 29-04-13 1:10
Primer fw 10 µM	0.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM	0.5 µl DelH_f2_KpnI_rev 10 µM
Q5 Ready Mix	10 µl
ddH₂O	7 µl

Cycles	Temperature [°C]	Time
1	98	10 s
12	98	1 s
	66 decrease by 0.5	5 s
	72	2:30 min
18	98	1 s
	66	5 s
	72	2:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 8 Kb, Loaded 1 µl of PCR

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!

Amplification of Backbone pSB6A1-AraC-lacZ

PCR Conditions BB.W9.A

Reagent	Backbone
Template	1 µl Backbone (pSB6A1-AraC-lacZ c = 24 ng/µl)
Primer fw 10 µM	0.5 µl AraCbb_KpnI_fw 100 µM
Primer rev 10 µM	0.5 µl AraCbbPacI_rev2 100 µM
Phusion Ready Mix	25 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time
1	98	30 s
35	98	15 s
	66	5 s
	72	3:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.11 Gel of amplified fragments of DelH and BB(loaded 1 µL)
l1:2log ladder, l5: F1b, l6: F2, l7: BB
no product

No band visible, amplification failed.

=> Why could we not reproduce the previous amplification?

PCR Conditions BB.W9.B

Reagent	BB
Template	1 µl Backbone (pSB6A1-AraC-lacZ c = 24 ng/µl)
Primer fw 10 µM	0.5 µl AraCbb_KpnI_fw 10 µM
Primer rev 10 µM	0.5 µl AraCbbPacI_rev2 10 µM
Phusion Ready Mix	25 µl
ddH₂O	23 µl

Cycles	Temperature [°C]	Time
1	98	30 s
12	98	15 s
	66 decrease by 0.5	15 s
	72	3:30 min
18	98	15 s
	66	15 s
	72	3:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig. 9.12 Gel of amplified DelH-fragments and BB l1:2log, l2: F1b,l3:F2, l4:BB

Unexpected band at 2.2 Kb

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.

PCR Conditions BB.W9.C

Reagent	DelH 2
Template	1 µl pSB6A1-AraC-LacZ digested and purified 1:10
Primer fw 10 µM	0.5 µl AraCbb_KpnI_fw 10 µM
Primer rev 10 µM	0.5 µl AraCbbPacI_rev2 10 µM
Phusion Flash Ready Mix	10 µl
ddH₂O	7 µl

Cycles	Temperature [°C]	Time
1	98	10 s
12	98	1 s
	66 decrease by 0.5	5 s
	72	2:30 min
18	98	1 s
	66	5 s
	72	2:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l4: BB shows no expected band

Gel does not show expected fragment.

=> Make sure right template was used. Is the restricted and purified fragment suitable?

PCR Conditions BB.W9.D

Reagent	DelH 2
Template	1 µl pSB6A1-AraC-LacZ digested and purified 1:10
Primer fw 10 µM	0.5 µl AraCbb_KpnI_fw 10 µM
Primer rev 10 µM	0.5 µl AraCbbPacI_rev2 10 µM
Q5 Ready Mix	10 µl
ddH₂O	7 µl

Cycles	Temperature [°C]	Time
1	98	10 s
12	98	1 s
	66 decrease by 0.5	5 s
	72	2:30 min
18	98	1 s
	66	5 s
	72	2:30 min
1	72	10 min
1	4	inf

Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!

@@ Line 1: / Line 1: @@
-==24-06 - 30-06-13 ==
+== 24-06 - 30-06-13 ==
 ===Characterization of DelH plasmid 19-06===
 ====SDS Page====
@@ Line 11: / Line 11: @@
 * Washed in ddH<sub>2</sub>O multiple times
 ====Result====
-[[File:CIMG1018.JPG|thumb|right|'''Fig.9.1''' SDS PAGE <br> ''L1:'' Ladder ''L2:'' 6 µl of Konrad’s ''E. coli'' ''L3:'' 6 µl DelH colony 4 ''L4:'' 6 µl DelH colony 6 ''L5:'' 9 µl of Konrad’s ''E. coli'' ''L6:'' 9 µl DelH colony 4 ''L7:'' 9 µl DelH colony 6 ''L8:'' 15 µl of Konrad’s ''E. coli'' ''L9:'' 15 µl DelH colony 4 ''L10:'' 15 µl DelH colony 6 ''L11:'' empty ''L12:'' Ladder  ]]
+[[File:Heidelberg_CIMG1018.JPG|thumb|right|'''Fig.9.1''' SDS PAGE <br> ''L1:'' Ladder ''L2:'' 6 µl of Konrad’s ''E. coli'' ''L3:'' 6 µl DelH colony 4 ''L4:'' 6 µl DelH colony 6 ''L5:'' 9 µl of Konrad’s ''E. coli'' ''L6:'' 9 µl DelH colony 4 ''L7:'' 9 µl DelH colony 6 ''L8:'' 15 µl of Konrad’s ''E. coli'' ''L9:'' 15 µl DelH colony 4 ''L10:'' 15 µl DelH colony 6 ''L11:'' empty ''L12:'' Ladder  ]]
 <div style="clear:both"></div>
 None clear band at ~600 kDa visible. Maybe in highest amounts, but not reliable.
@@ Line 38: / Line 38: @@
 Expected band pattern: 5 kp, 7.3 kp, 13 kp
 <br/>
-[[File:20130626 2log test digested.png|200px|thumb|right|'''Fig.9.2''' Gel of amplified fragments (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:''test digested DelH fragment <br> ''l2:'' no visible band ]]
+[[File:Heidelberg_20130626 2log test digested.png|200px|thumb|right|'''Fig.9.2''' Gel of amplified fragments (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:''test digested DelH fragment <br> ''l2:'' no visible band ]]
 <div style="clear:both"></div>
 No clear bands visible.
@@ Line 90: / Line 90: @@
 Expected bands: BB (7.3 Kb), DelH F2 (8 Kb), F1a + F1b (10 Kb)
 <br/>
-[[File:20130627 2log test digested.png|200px|thumb|right|'''Fig.9.3''' miniprep resitriction digested (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:'' DelH-F1a <br> ''l2:'' DelH-F1a shows specific band = cut out ]]
+[[File:Heidelberg_20130627 2log test digested.png|200px|thumb|right|'''Fig.9.3''' miniprep resitriction digested (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:'' DelH-F1a <br> ''l2:'' DelH-F1a shows specific band = cut out ]]
 <div style="clear:both"></div>
 Unexpected bands at 3 Kb, but not desired ones.
@@ Line 133: / Line 133: @@
 Expected band: 5 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130627 2log F1b.png|200px|thumb|right|'''Fig.9.4''' gel of amplified DelH 1b - fragment (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:'' DelH-F1b <br> ''l2:'' DelH-F1b shows no band]]
+[[File:Heidelberg_20130627 2log F1b.png|200px|thumb|right|'''Fig.9.4''' gel of amplified DelH 1b - fragment (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:'' DelH-F1b <br> ''l2:'' DelH-F1b shows no band]]
 <div style="clear:both"></div>
 No correct band visible.
@@ Line 174: / Line 174: @@
 Expected band: 5 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130627 2log F1bREP.png|200px|thumb|right|'''Fig.9.5''' gel of amplified DelH-1b-fragment (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:'' DelH-F1b <br> ''l2:'' DelH-F1b shows unexpected band  at 2.3 Kb]]
+[[File:Heidelberg_20130627 2log F1bREP.png|200px|thumb|right|'''Fig.9.5''' gel of amplified DelH-1b-fragment (loaded 20 µL) <br> ''l1:'' 2 log ladder, ''l2:'' DelH-F1b <br> ''l2:'' DelH-F1b shows unexpected band  at 2.3 Kb]]
 <div style="clear:both"></div>
 Again, expected band not there.
@@ Line 215: / Line 215: @@
 Expected band: 5 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130628 2log F1b F2 BB.png|200px|thumb|right|'''Fig.9.6''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l2: F1a shows specific band at 5 Kb]]
+[[File:Heidelberg_20130628 2log F1b F2 BB.png|200px|thumb|right|'''Fig.9.6''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l2: F1a shows specific band at 5 Kb]]
-  [[File:20130628 2log F1b F2 BB cut.png|200px|thumb|right|'''Fig.9.7''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l2: F1a shows specific band at 5 Kb = was cut out]]
+  [[File:Heidelberg_20130628 2log F1b F2 BB cut.png|200px|thumb|right|'''Fig.9.7''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l2: F1a shows specific band at 5 Kb = was cut out]]
 <div style="clear:both"></div>
 Highly specific band at 5 Kb, as well as additional smaller ones.
 :=> Band was cut. Run gel with remaining sample for gel extraction.
-[[File:20130628 2log F1b F2 flash all.png|200px|thumb|right|'''Fig.9.8''' gel of amplified fragments (loaded 20 µL) <br> ''l1-3:''F1b, ''l4:'' 2log ladder, ''l5-7:'' BB  <br> BB was not amplified]]
+[[File:Heidelberg_20130628 2log F1b F2 flash all.png|200px|thumb|right|'''Fig.9.8''' gel of amplified fragments (loaded 20 µL) <br> ''l1-3:''F1b, ''l4:'' 2log ladder, ''l5-7:'' BB  <br> BB was not amplified]]
-  [[File:20130628 2log F1b F2 flash all cut.png|200px|thumb|right|'''Fig.9.9''' gel of amplified fragments (loaded 20 µL) <br> ''l1-3:''F1b, ''l4:'' 2log ladder, ''l5-7:'' BB  <br> BB was not amplified]]
+  [[File:Heidelberg_20130628 2log F1b F2 flash all cut.png|200px|thumb|right|'''Fig.9.9''' gel of amplified fragments (loaded 20 µL) <br> ''l1-3:''F1b, ''l4:'' 2log ladder, ''l5-7:'' BB  <br> BB was not amplified]]
 <div style="clear:both"></div>
 <br/>
@@ Line 261: / Line 261: @@
 Expected band: DelH F1b 5 Kb
 <br/>
-[[File:20130628 2log genomicintegration DelH-1 2 BB.png|200px|thumb|right|'''Fig.9.10''' gel of amplified fragments using Q5 (loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' fragment 1b, ''l6:'' fragment 2, ''l7:'' BB  <br> no product]]
+[[File:Heidelberg_20130628 2log genomicintegration DelH-1 2 BB.png|200px|thumb|right|'''Fig.9.10''' gel of amplified fragments using Q5 (loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' fragment 1b, ''l6:'' fragment 2, ''l7:'' BB  <br> no product]]
 <div style="clear:both"></div>
 Neither of the PCRs yield any product.
@@ Line 304: / Line 304: @@
 Expected band: 8 Kb
 <br/>
-[[File:20130627 2log DelH-1b 2 BB 1pcr.png|200px|thumb|right|'''Fig.9.11''' gel of amplified fragments (loaded 1 µL) <br>''l1:'' 2log ladder, ''l2:''F1b,  ''l3:'' F2, ''l4:'' BB  <br> shows band at 3 Kb]]
+[[File:Heidelberg_20130627 2log DelH-1b 2 BB 1pcr.png|200px|thumb|right|'''Fig.9.11''' gel of amplified fragments (loaded 1 µL) <br>''l1:'' 2log ladder, ''l2:''F1b,  ''l3:'' F2, ''l4:'' BB  <br> shows band at 3 Kb]]
 <div style="clear:both"></div>
 Unexpected bands at 3 Kb in lanes of DelH 2
@@ Line 352: / Line 352: @@
 Expected band: 8 Kb
 <br/>
-[[File:20130627 2log DelH-1b 2 BB.png|200px|thumb|right|'''Fig.9.12''' gel of amplified fragments (loaded 1 µL) <br> ''l1:''2log, ''l2:'' F1b, ''l3:'' F2, ''l4:'' BB  <br> F2 shows no specific band]]
+[[File:Heidelberg_20130627 2log DelH-1b 2 BB.png|200px|thumb|right|'''Fig.9.12''' gel of amplified fragments (loaded 1 µL) <br> ''l1:''2log, ''l2:'' F1b, ''l3:'' F2, ''l4:'' BB  <br> F2 shows no specific band]]
 <div style="clear:both"></div>
 Unexpected bands at 1.5 Kb, 2.2 Kb, 4 Kb and 6 Kb. Desired 8 Kb band is present but hardly visible.
@@ Line 400: / Line 400: @@
 Expected band: 8 Kb
 <br/>
-[[File:20130628 2log F1b F2 BB.png|200px|thumb|right|'''Fig.9.6''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l3: F2 shows specific band at 8 Kb and other ones]]
+[[File:Heidelberg_20130628 2log F1b F2 BB.png|200px|thumb|right|'''Fig.9.6''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l3: F2 shows specific band at 8 Kb and other ones]]
-[[File:20130628 2log F1b F2 BB cut.png|200px|thumb|right|'''Fig.9.7''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l2: F1a shows specific band at 8 Kb = was cut out]]
+[[File:Heidelberg_20130628 2log F1b F2 BB cut.png|200px|thumb|right|'''Fig.9.7''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l2: F1a shows specific band at 8 Kb = was cut out]]
 <div style="clear:both"></div>
 F2 shows specific band at 8 Kb and additional ones.
 :=> Band was cut. Run gel with remaining sample for gel extraction.
 <div style="clear:both"></div>
-[[File:20130628 2log F1b F2 flash all.png|200px|thumb|right|'''Fig.9.8''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2 <br> l3: F2 shows specific band at 8 Kb and other ones]]
+[[File:Heidelberg_20130628 2log F1b F2 flash all.png|200px|thumb|right|'''Fig.9.8''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2 <br> l3: F2 shows specific band at 8 Kb and other ones]]
-[[File:20130628 2log F1b F2 flash all cut.png|200px|thumb|right|'''Fig.9.9''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2  <br> l3: F2 shows specific band at 8 Kb and other ones]]
+[[File:Heidelberg_20130628 2log F1b F2 flash all cut.png|200px|thumb|right|'''Fig.9.9''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2  <br> l3: F2 shows specific band at 8 Kb and other ones]]
 <div style="clear:both"></div>
 <br/>
@@ Line 453: / Line 453: @@
 Expected band: 8 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130628 2log genomicintegration DelH-1 2 BB.png|200px|thumb|right|'''Fig.9.10''' gel of amplified fragments using Q5 (loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' fragment 1b, ''l6:'' fragment 2, ''l7:'' BB  <br> no product]]
+[[File:Heidelberg_20130628 2log genomicintegration DelH-1 2 BB.png|200px|thumb|right|'''Fig.9.10''' gel of amplified fragments using Q5 (loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' fragment 1b, ''l6:'' fragment 2, ''l7:'' BB  <br> no product]]
 <div style="clear:both"></div>
 Neither of the PCRs yield any product.
@@ Line 497: / Line 497: @@
 Expected band: 7.3 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130627 2log DelH-1b 2 BB 1pcr.png|200px|thumb|right|'''Fig.9.11''' Gel of amplified fragments of DelH and BB(loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' F1b, ''l6:'' F2, ''l7:'' BB  <br> no product]]
+[[File:Heidelberg_20130627 2log DelH-1b 2 BB 1pcr.png|200px|thumb|right|'''Fig.9.11''' Gel of amplified fragments of DelH and BB(loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' F1b, ''l6:'' F2, ''l7:'' BB  <br> no product]]
 <div style="clear:both"></div>
 No band visible, amplification failed.
@@ Line 546: / Line 546: @@
 Expected band: 7.3 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130627 2log DelH-1b 2 BB.png|200px|thumb|'''Fig. 9.12''' Gel of amplified DelH-fragments and BB ''l1:''2log, ''l2:'' F1b,''l3:''F2, ''l4:''BB]]
+[[File:Heidelberg_20130627 2log DelH-1b 2 BB.png|200px|thumb|'''Fig. 9.12''' Gel of amplified DelH-fragments and BB ''l1:''2log, ''l2:'' F1b,''l3:''F2, ''l4:''BB]]
 <div style="clear:both"></div>
 Unexpected band at 2.2 Kb
@@ Line 594: / Line 594: @@
 Expected band: 7.3 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130628 2log F1b F2 BB.png|200px|thumb|right|'''Fig.9.6''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l4: BB shows no expected band]]
+[[File:Heidelberg_20130628 2log F1b F2 BB.png|200px|thumb|right|'''Fig.9.6''' Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL) <br> ''l1:''2log ladder, ''l2:'' F1a, ''l3:''F2, ''l4:'' BB  <br> l4: BB shows no expected band]]
 <div style="clear:both"></div>
 Gel does not show expected fragment.
@@ Line 641: / Line 641: @@
 Expected band: 7.3 Kb, Loaded 1 µl of PCR
 <br/>
-[[File:20130628 2log genomicintegration DelH-1 2 BB.png|200px|thumb|right|'''Fig.9.10''' gel of amplified fragments using Q5 (loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' fragment 1b, ''l6:'' fragment 2, ''l7:'' BB  <br> no product]]
+[[File:Heidelberg_20130628 2log genomicintegration DelH-1 2 BB.png|200px|thumb|right|'''Fig.9.10''' gel of amplified fragments using Q5 (loaded 1 µL) <br> ''l1:''2log ladder, ''l5:'' fragment 1b, ''l6:'' fragment 2, ''l7:'' BB  <br> no product]]
 <div style="clear:both"></div>
 Neither of the PCRs yield any product.
 :=> Therefore, Q5 is no option for us!
 <br/>

Team:Heidelberg/Templates/DelH week9

From 2013.igem.org

Revision as of 20:55, 1 October 2013

Contents

24-06 - 30-06-13

Characterization of DelH plasmid 19-06

SDS Page

Result

Mini Prep

Test Restriction Digest

Result

DNA Concentration

Repetion of Mini Prep

DNA Concentration

Test Restriction Digest

Result

Amplification of DelH F1b

PCR Conditions F1b.W9.A

Result

PCR Conditions F1b.W9.B

Result

PCR Conditions F1b.W9.C

Result

PCR Conditions F1b.W9.D

Result

Amplification of DelH F2

PCR Conditions F2.W9.A

Result

PCR Conditions F2.W9.B

Result

PCR Conditions F2.W9.C

Result

PCR Conditions F2.W9.D

Result

Amplification of Backbone pSB6A1-AraC-lacZ

PCR Conditions BB.W9.A

Result

PCR Conditions BB.W9.B

Result

PCR Conditions BB.W9.C

Result

PCR Conditions BB.W9.D

Result