Team:Heidelberg/Templates/DelH week16

From 2013.igem.org

Contents

12-08 - 18-08-13

Increasing Yield of DelH Fragments

PCR Conditions G0.G1/2a.W16.A

Reagent G0 G0G0G1/2a G1/2a
Expected length [Kb] 18 18 18 13.083 13.083
Named G0 G0 G0 1 1
Template Picked colony Picked colony Picked colony Picked colony Picked colony
Primer fw 10 µM 1 µl short2 1 µl short2 1 µl short2 1 µl HM07 1 µl HM07
Primer rev 10 µM 1 µl HM08 1 µl HM08 1 µl HM08 1 µl HM06 1 µl HM06
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5 µl 5 µl 5 µl
DMSO 1 µl 1 µl 1 µl 1 µl 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start used

Result

Expected bands: 18 Kb (G0), 13 Kb (G1/2a)

Fig.16.1 gel of amplified DelH-fragment(loaded 20 µL of PCR)
l1-3: G0 (complete DelH-fragment with DN11 & HM08), l4-5: G1/2b (3/4 of the DelH fragment amplified with DN11 & HM06), l6: 1kb+ ladder
l2 shows the expected band at 18 Kb. l4-5 showed expected band at 13 Kb. All three bands were cut out.

Gel shows expected bands.

=> They were cut and gel isolated.


Generation of Plasmid DelH 11-08

Colony-PCR CP.W16.A

Reagent Cells + 1 µl Gibson Assembly Cells + 14 µl Gibson Assembly Cells + 14 µl Gibson Assembly
(ONLY Backbone) 		Cells + 14 µl Gibson Assembly
(ONLY Backbone)
Expected length [bp] 663 663 - -
Named D1 D14 B14(1) B14(2)
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.16.2 gel of screening PCRs with DN07 & VF2 primer (loaded 20 µL of PCR)
l1: 2log ladder, l2: colony PCR of 1 µl Gibson transformed,l3: colony PCR of 14 µl Gibson transformed,l4-5: 2 colony PCRs of BB - 14 µl Gibson transformed, l6: 2log ladder
none of the colonies is positive, of course the BB shows not the expected band of 663 bp (for screening). The first colony shows a band at ~1.7 kb.

None of the colonies is positive, of course the BB shows not the expected band of 663 bp (for screening). The first colony shows a band at ~1.7 kb.

=> None of the colonies harbours DelH plasmid.


Colony-PCR CP.W16.B

Reagent Cells + 10 µl Gibson Assembly
pink 		Cells + 10 µl Gibson Assembly
pink 		Cells + 10 µl Gibson Assembly
white 		Cells + 10 µl Gibson Assembly
white
Expected length [bp] 663 663 663 663
Named A B C D
Primer fw 10 µM 2 µl VF2 2 µl VF2 2 µl VF2 2 µl VF2
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl 10 µl 10 µl
ddH2O 6 µl 6 µl 6 µl 6 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5 °C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.16.3 Gel of colony PCR (loaded 10 µL)
l1: 2log ladder, l2-13 screening colonies A-D


Testing of Screening Primers

To test the reverse screening primer DN07 we amplify with the DreamTaqPolymerase and DN11 primer. As template, we use the complete DelH (18 Kb) and the first 3/4 of DelH (14 Kb).

Reagent 1 µl G0 of 23-07 1 µl G1/2a of 23-07
Expected length [bp] 323 323
Named 0 1
Primer fw 10 µM 2 µl DN11 2 µl DN11
Primer rev 10 µM 2 µl Screen_delH_rev 2 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl
ddH2O 5 µl 5 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 30
18 95 60
65 (touchdown -0.5°C) 30
72 30
1 12 inf

Result

Expected length= 323 bp for positive clones

Fig.16.4 Gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 screening G0, lane 3 screening G1/2a

Gel shows expected bands.

=> Both PCRs were positive. Proof of functionality of DN07 screening primer and that both fragments are the expected DelH fragments.


Amplification of DelH G0

Gradient PCR Conditions G0.W16.B

To improve the PCR of the fragment with the length of 18 Kb, we run a gradient PCR between 68°C and 65°C.

Reagent G0 G0G0G0
Expected length [Kb] 18 18 18 18
Named G0 1 G0 2 G0 3 G0 4
Template Picked colony Picked colony Picked colony Picked colony
Primer fw 10 µM 2 µl short2 2 µl short2 2 µl short2 2 µl short2
Primer rev 10 µM 2 µl HM08 2 µl HM08 2 µl HM08 2 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5 µl 5 µl
DMSO 1 µl 1 µl 1 µl 1 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 98 5
30 98 1
68 - 65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.16.5 amplified fragment of DelH (loaded 20 µL of PCR)
l1:1kb+ ladder, l2-5: G0 = complete DelH, l6-9: G1/2a, l10:1kb+ ladder
l2-5: show not the expected band at 18Kb. l6-9 show the expected band at 13.5 Kb and was cut out. But it shows also other unspecific bands.

Gel does not show expected band.

=> Further optimize PCR conditions.


PCR Conditions G0.W16.C

Because the amplification of DelH GO didn't work yesterday, we try it again with one picked colony and 1 µl of a glycerol stock at a constant annealing temperature of 65°C. 65°C was the temperature on which it worked the last time.

Reagent G0 G0
Expected length [Kb] 18 18
Named G0 1 G0 2
Template Picked colony SPH1 1 µl of glycerol stock SPH1
Primer fw 10 µM 2 µl short2 2 µl short2
Primer rev 10 µM 2 µl HM08 2 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl
ddH2O 5 µl 4 µl
DMSO 1 µl 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.16.6 gel of amplified DelH G0 fragment (loaded 20 µL of PCR)
l1: G0 amplified from picked colony, l2: G0 amplified from glycerol stock, l3: 1 kb+ ladder
l3 shows expected band at 18 kb and was cut

Gel shows expected band.

=> It was cut and gel extracted. Also, we will run again 3 samples with the same conditions.


PCR Conditions G0.W16.D

Reagent G0 G0 G0
Expected length [Kb] 18 18 18
Named G0 1 G0 2 G0 2
Template 1 µl of glycerol stock SPH1 1 µl of glycerol stock SPH1 1 µl of glycerol stock SPH1
Primer fw 10 µM 2 µl short2 2 µl short2 2 µl short2
Primer rev 10 µM 2 µl HM08 2 µl HM08 2 µl HM08
Phusion Flash Ready Mix 10 µl 10 µl 10 µl
ddH2O 4 µl 4 µl 4 µl
DMSO 1 µl 1 µl 1 µl
Cycles Temperature DelH-G0 [°C] Time [s]
1 98 5
30 98 1
65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 18 Kb

Fig.16.7 gel of amplified DelH G0 in same conditions (loaded 20 µL of PCR)
l1: 1kb+ ladder, l2: G0 amplified with DN11 & HM08, l3: G0, l4: G0
l2-4show the expected band at 18 Kb and was cut

Gel shows expected fragments.

=> They were cut and gel extracted.


Amplification of DelH G1/2a

Gradient PCR Conditions G1/2a.W16.B

To improve the PCR of the fragment with the length of 18 Kb, we run a gradient PCR between 68°C and 65°C.

Reagent G1/2a G1/2a G1/2a G1/2a
Expected length [Kb] 13.083 13.083 13.083 13.083
Named 1 1 1 2 1 3 1 4
Template Picked colony Picked colony Picked colony Picked colony
Primer fw 10 µM 2 µl short2 2 µl short2 2 µl short2 2 µl short2
Primer rev 10 µM 2 µl HM06 2 µl HM06 2 µl HM06 2 µl HM06
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl
ddH2O 5 µl 5 µl 5 µl 5 µl
DMSO 1 µl 1 µl 1 µl 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
68 - 65 5
72 4:45 min
1 72 10 min
1 12 inf
  • Lid preheated at 98°C
  • No hot start

Result

Expected band: 13 Kb

Fig.16.5 amplified fragment of DelH (loaded 20 µL of PCR)
l1:1kb+ ladder, l2-5: G0 = complete DelH, l6-9: G1/2a, l10:1kb+ ladder
l2-5: show not the expected band at 18Kb. l6-9 show the expected band at 13.5 Kb and was cut out. But it shows also other unspecific bands.

Gel shows expected band of G1/2a, but also additional uspecific bands.

=> Fragment was cut and gel extracted.


Generation of Plasmid DelH 17-08

Electroporation

Remaining Gibson mix from 01.08 was electroporated.

Colony-PCR CP.W16.B

5 colony per PCR tube (but in different LB Amp Eppis)

Reagent Electroporation from 17-08 10 µl Electroporation from 17-08 100 µl
Expected length [bp] 663 663
Named 1 - 2 3 - 12
Template 2 colonies of 10 µl 10 colonies of 100 µl
Primer fw 10 µM 1 µl VF2 1 µl VF2
Primer rev 10 µM 1 µl Screen_delH_rev 1 µl Screen_delH_rev
Dream-Taq Polymerase (2x) 10 µl 10 µl
ddH2O 8 µl 8 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 (touchdown -0.5°C) 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.16.8 gel of screening minipreped colonies 1&2 from plate with 10 µl and colonies 3-12 from plate with 100 ml Gibson assembled construct with mRFP (loaded 20 µL of PCR)
l1: 2log ladder, l2: 1, l3: 2, l4: 3, l5: 4, l6: 5, l7: 6, l8: 7, l9: 8, l10: 9, l11: 10, l12: 11, l13: 12
l4-13 show expected band = positive?

Majority of colonies show expected band.

=> Grow ON, perform mini prep and test digests as well as PCRs of fragments from isolated DNA.


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