Team:Heidelberg/Tyrocidine week17 interms

From 2013.igem.org

Contents

Amplifications

In the course of this project, we'll have to amplify the following fragments: Fragments for Tyrocidine-Indigoidine-Fusion

For reasons of clarity, the gel-pictures obtained by PCR are shown here in this gallery:

Heidelberg A-I 1st PCR.png Heidelberg AEG 2nd PCR.png Heidelberg A-I 3rd PCR.png Heidelberg D 4th PCR.png

Comment on the second File:Heidelberg_

The normal gel-image before the cut out was not properly saved on the PC that is connected to the camera. The bands that were visible (one can estimate them by the cut-out) were at about 2.4 to 2.5 kbp, which is the expected size of the backbone

Fragment A

A

File:Heidelberg ...

what µl
pSB1C3 1
IK21 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 5
35 98 1
66 5
72 40
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to a too low dilution of Medi-Prep and hence too much template. For optimization use less template and take it from a more diluted sample.

B

what µl
pSB1C3 0.5
IK21 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
35 98 1
66 5
72 40
1 72 300
1 10 inf

Results

Still the gel is overloaded, though less template was used. For the next time, reduce cycle repetitin from 35 to 30 and increase annealing temperature by 1°C

C

what µl
pSB1C3 0.5
IK21 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
30 98 1
67 5
72 40
1 72 300
1 10 inf

Results


Fragment B

A

File:Heidelberg ...

what µl
B. parabrevis 1
IK16 2
PW17 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
35 98 1
65 5
72 40
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

what µl
B. parabrevis 1
IK16 2
PW17 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
30 98 1
66 5
72 40
1 72 300
1 10 inf

Results


Fragment C

A

File:Heidelberg ...

what µl
B. parabrevis 1
PW18 2
PW14 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
35 98 1
65 5
72 40
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

what µl
B. parabrevis 1
PW18 2
PW14 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
30 98 1
66 5
72 40
1 72 300
1 10 inf

Results

Fragment D

A

For obtaining more product and optimizing the conditions simultaneously, a gradient PCR with touch-down was carried out. 4 wells were used at 62°C, 61.4°C, 60.6°C and 60°C for touchdown, with steady temperatures at 59°C, 58.4°C, 57.6°C and 57°C.

what µl
indC (RB37, RB38) 0.5
PW15 2
PW16 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
12 98 5
62 ↓ 0.5 to 60 ↓ 0.5 5
72 100
23 98 5
59 to 57 10
72 100
1 72 600
1 10 inf

Results

Fragment E

A

File:Heidelberg ...

what µl
pSB1C3 1
PW25 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 5
35 98 1
66 5
72 40
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to a too low dilution of Medi-Prep and hence too much template. For optimization use less template and take it from a more diluted sample.

B

what µl
pSB1C3 0.5
PW25 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
35 98 1
66 5
72 40
1 72 300
1 10 inf

Result

Still the gel is overloaded, though less template was used. For the next time, reduce cycle repetitin from 35 to 30 and increase annealing temperature by 1°C

C

what µl
pSB1C3 0.5
PW25 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
30 98 1
67 5
72 40
1 72 300
1 10 inf

Results


Fragment F

A

File:Heidelberg ...

what µl
B. parabrevis 1
PW24 2
PW14 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
35 98 1
59 5
72 60
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

what µl
B. parabrevis 1
PW24 2
PW14 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
30 98 1
61 5
72 60
1 72 300
1 10 inf

Results


Fragment G

A

File:Heidelberg ...

what µl
pSB1C3 1
PW22 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 5
35 98 1
66 5
72 40
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to a too low dilution of Medi-Prep and hence too much template. For optimization use less template and take it from a more diluted sample.

B

what µl
pSB1C3 0.5
PW22 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
35 98 1
66 5
72 40
1 72 300
1 10 inf

Results

Still the gel is overloaded, though less template was used. For the next time, reduce cycle repetitin from 35 to 30 and increase annealing temperature by 1°C

C

what µl
pSB1C3 0.5
PW22 2
PW23 2
Phusion Flash 2x Master mix 10
ddH20 5.5
Cycles temperature [°C] Time [s]
1 98 5
30 98 1
67 5
72 40
1 72 300
1 10 inf

Results


Fragment H

A

File:Heidelberg ...

what µl
B. parabrevis 1
PW19 2
PW20 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
35 98 1
59 5
72 60
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

what µl
B. parabrevis 1
PW19 2
PW20 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
30 98 1
61 5
72 60
1 72 300
1 10 inf

Results


Fragment I

A

File:Heidelberg ...

what µl
B. parabrevis 1
PW21 2
PW14 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
35 98 1
59 5
72 60
1 72 300
1 10 inf

Results

The gel was overloaded, which might be due to the polymerase, as Q5 was usually used in previous samples and bright bands are visible for all fragments. For optimization, the annealing-temperature could be increased by 1 - 2°C and cycle repetition may be reduced to 30 cycles.

B

what µl
B. parabrevis 1
PW21 2
PW14 2
Phusion Flash 2x Master mix 10
ddH20 5
Cycles temperature [°C] Time [s]
1 98 120
30 98 1
61 5
72 60
1 72 300
1 10 inf

Results


Quantification-gels

For the further work, the amount of DNA available has to be quantified. We decided to do this by a quantification gel in order to check the right sizes of the constructs. The fragments A, E and G from the first PCR were not used due to the big smear and the double-bands, instead the bands obtained in the second PCR were taken for the quantification.

Heidelberg A-I 1st quant.png

The bands did not have a definite size, hence they are not useful to work with due to unwanted byproducts that are obviously present. After the optimizations regarding annealing-temperature and cycle-repetition, the following bands were obtained during quantification

Heidelberg A-I 2nd quant.png Heidelberg D 3rd quant.png

Here, the bands have a definded size and a sufficient quantity to work with. The quantification for fragment D was carried out later, as the amplification of those fragments happened later.