Team:Heidelberg/Templates/DelH week10

From 2013.igem.org

Contents

01-07 - 07-07-13

Amplification of DelH F1b

PCR Conditions F1b.W10.A

Reagent DelH F1b
Template 1 µl DelH 1Fb Fragment
Primer fw 10 µM 2.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM 2.5 µl DelH_f1_SalI_rev 10 µM
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 30
30 98 15
68 15
72 5 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 5 Kb

Fig.10.1 gel of amplified fragments (loaded 1 µL of PCR)
l1: 2log ladder, l2: F1b,l3: F2, l4: BB
no bands

No correct bands visible.

=> Phusion is no option for us, not even for re-PCRs.


PCR Conditions F1b.W10.B

Reagent DelH F1b
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl DelH_EcoRI_fw 10 µM
Primer rev 10 µM 2.5 µl DelH_f1_SalI_rev 10 µM
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
68 5
72 180
1 72 5 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 5 Kb

Fig.10.2 gel of amplified DelH fragments (loaded 50 µL of PCR)
l1-2: F1b, l3: 2log,l4-5:fragment 2 of DelH, l6-7: pSB6A1
l1: F1b shows no band
=> F1b was not amplified.


Amplification of DelH F2

PCR Conditions F2.W10.A

Reagent DelH F2
Template 1 µl DelH 2 PCR product
Primer fw 10 µM 2.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM 2.5 µl DelH_f2_KpnI_rev 10 µM
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 15
66 decrease by 0.5 15
72 8 min
12 98 15
66 15 s
72 8 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.10.1 gel of amplified fragments (loaded 50 µL of PCR)
l1: F1b, l2: 2, l3: pSB6A1
l2: F2 shows no band

Gel does not show correct band.

=> Phusion is no option for us, not even for re-PCRs.


PCR Conditions F2.W10.B

Reagent DelH F2
Template 1 µl D. acidovorans glycerol stock
Primer fw 10 µM 2.5 µl DelH_f2_SalI_fw 10 µM
Primer rev 10 µM 2.5 µl DelH_f2_KpnI_rev 10 µM
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66 decrease by 0.5 5
72 2:30 min
12 98 1
66 5
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 8 Kb, Loaded 50 µl of PCR

Fig.10.2 gel of amplified fragments (loaded 50 µL of PCR)
l1-2: F1b, l3: 2log, l4-5: 2,l6-7 pSB6A1-AraC-lacZ
l1: F1b shows no band
=> F2 was not amplified.


Amplification of Backbone

PCR Conditions BB.W10.A

Reagent Backbone
Template 1 µl psB6A1+AraC+Lacz 1:10
Primer fw 10 µM 2.5 µl AraCbb_KpnI_fw
Primer rev 10 µM 2.5 µl AraCbbPacI_rev2
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
66 decrease by 0.5 15
72 8 min
12 98 15
66 15
72 8 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.10.1 gel of amplified fragments (loaded 1 µL of PCR)
l1: 2log ladder, l2: F1b,l3: F2, l4: BB
no bands

No product.

=> Phusion is no option for us, not even for re-PCRs.


PCR Conditions BB.W10.B

Reagent Backbone
Template 1 µl psB6A1+AraC+Lacz 1:10
Primer fw 10 µM 2.5 µl AraCbb_KpnI_fw
Primer rev 10 µM 2.5 µl AraCbbPacI_rev2
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66 decrease by 0.5 5
72 2:30 min
12 98 1
66 5
72 2:30 min
1 72 10 min
1 4 inf
  • Using hot start at 98°C

Result

Expected band: 7.3 Kb

Fig.10.2 gel of amplified fragments (loaded 50 µL of PCR)
l1-2: F1b, l3: 2log,l4-5:fragment 2 of DelH, l6-7: pSB6A1
l1: F1b shows no band
=> BB was not amplified completely. It seems that there is no insert present or the conditions were not optimal. Repeat the PCR with other conditions.


PCR Conditions BB.W10.C

Reagent Backbone
Template 1 µl psB6A1+AraC+Lacz 1:10
Primer fw 10 µM 2.5 µl AraCbb_KpnI_fw
Primer rev 10 µM 2.5 µl AraCbbPacI_rev2
Phusion Ready Mix 25 µl
ddH2O 19 µl
Cycles-PCR Temperature [°C] Time [s]
1 98 10
30 98 1
66 5
72 2:15 min
1 72 180
1 4 inf

Result

Expected bands: 7.3 Kb, Loaded 50 µl of PCR

Fig.10.4 gel of amplified fragments (loaded 50 µL of PCR)
l1: F1b, l2: 2, l3: pSB6A1
specific bands at 7,3 Kb was cut out
Fig.10.3 gel of amplified fragments (loaded 50 µL of PCR)
l1: F1b, l2: 2, l3: pSB6A1
band at specific 7,3 Kb, but also unspecific ones

Expected band is there, but also unspecific ones.

=> Correct band was cut and gel extracted.


Generation of DelH Plasmid

Current Status

Fragment Concentration [ng/µl] Digested and Purified
F1a 267 check
F1b 9.8 -
F2 11 (gel extracted) and 369 (PCR purified) -
pSB6A1-AraC-lacZ 53.5 and 38.5 -

Restriction Digest of Fragments

Fragment Amount for Digest [ng] Enzymes [µl] Buffer [µl]
F1b 441

EcoRI-HF: 1
SalI-HF: 1

Cut smart 5.5
F2 495

KpnI: 1
SalI-HF: 1

Buffer 2.1: 5.5
pSB6A1-AraC-lacZ 1,599

KpnI: 1
PacI: 1

Buffer 1.1: 3.5
  • 1 h at 37°C

Isopropanol Purification

Fragment Concentration after Digest & Purification [ng/µl] Sequences
F1a 267

File:Heidelberg PCR(DelH) restricted(KpnI+PacI) sequence.fasta.txt

F1b 3.5 (Entire) Sequence of DelH
F2 1.8
pSB6A1-AraC-lacZ 7.3

File:Heidelberg PCR(pSB6A1-AraC-lacZ) digest.fasta.txt
File:Heidelberg PCR(pSB6A1-AraC-lacZ) sequence.fa.txt

Confirmation of DNA Concentration

Fragment Concentration [ng/µl] Date µl on gel Expected band size
F1a 261 19-06-13 1 µl 5 Kb
F1a 261 19-06-13 2 µl 5 Kb
F2 369 01-07-13 1 µl 8 Kb
Fig.10.5 concentration validation (loaded 1 µL of F1a & F2 PCR)
l1: F1a, l2: F2, l3: 2log
there are tiny bands showing the expected fragment for F2, F1a shows no band

Fragment F1a is not present, all others are fine.

=> Therefore, the next steps are:
1. Measurement of concentration = 40 ng/µl
2. PCR of the fragment F1a. As template, we use the purified PCR with the concentration of 40 ng/µl.

Restriction Digest of F2 and Backbone

Fragment Volume for digest [ng] Enzymes [µl] Buffer [µl]
2 40*369 = 14.76 µg

KpnI: 1
SalI-HF: 1

Buffer 2.1: 4.8
pSB6A1-AraC-lacZ 40*38.5 = 1.54 µg

KpnI: 1
PacI: 1

Buffer 1.1: 1.8
  • 1h at 37°C

Purification of Restriction Digest

Using nucleotide removal kit (Qiagen), eluted with 30 µl ddH2O.

Fragment Concentration [ng/µl] µl available
DelH F2 30 25 µl
pSB6A1-AraC-lacZ -11 25 µl


Amplification of DelH F1a

PCR Conditions F1a.W10.A

Reagent DelH F1a
Template 0.5 µl of DelH F1a digested Fragment 1:5 (= 8 ng)
Primer fw 10 µM 2.5 µl DelH_f1_PacI_fw
Primer rev 10 µM 2.5 µl DelH_EcoRI_rev
Phusion Flash Ready Mix 25 µl
ddH2O 19.5 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
66 5
72 120
1 72 10 min
1 4 inf

Result

Expected band: 5 Kb

Fig.10.6 gel of amplified fragments (loaded 20 µL)
l1: F1a ,l2: 2log ladder,l3:F1b
no band visible

Gel does not show any bands.

=> We conclude, that the fragments we expected to be F1a cannot be amplified by PCR. Thus, they were not amplified in the PCR used as template.


PCR Conditions F1a.W10.B

Reagent DelH F1a
Template 1 µl ‘‘D. acidovorans’’ glycerol stock
Primer fw 10 µM 2.5 µl DelH_f1_short2_fw
Primer rev 10 µM 2.5 µl DelH_EcoRI_rev
Phusion Flash Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
66 5
72 2:00 min
1 72 7 min
1 4 inf

Result

See Confirmation of DNA Concentration

Amplification of DelH F1b

PCR Conditions F1b.W10.C

Reagent DelH F1b
Template 1 µl DelH F1b digested Fragment
Primer fw 10 µM 2.5 µl DelH_EcoRI_fw
Primer rev 10 µM 2.5 µl DelH_f1_SalI_rev
Phusion Flash Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
68 5
72 120
1 72 10 min
1 4 inf

Result

Expected band: 5 Kb

Fig.10.6 gel of amplified fragments (loaded 20 µL)
l1: F1a ,l2: 2log ladder,l3:F1b
no band visible

There is no band visible.

=> We conclude, that the fragments we expected to be F1b cannot be amplified by PCR. Thus, they were not amplified in the PCR used as template.


Generation of DelH Plasmid

Confirmation of DNA Concentration

Fragment Concentration (by Nanodrop) µl on gel µl loading dye Digested and purified (V + A) Date (of PCR or Purification) Check on gel
F1a (PCR) - 1 9* 1x - 03-07-2013 (PCR) +
F1a 40 2 9* 1x V + A 19-06-2013 -
F1b 19 3 1* 6x V + A 19-06-2013 -
F1b 3.5 10 2* 6x V + A 02-07-2013 (V+A) -
F2 0.5 10 2* 6x V + A -
F2 30.5 2 10* 1x V + A (Nucleotide Removal) 03-07-2013 +
F2 (1:5 dilution of) 36.9 2 10* 1x - 02-07-2013 + (BackUP)
pSB6A1-AraC-lacZ 2.2 10 2* 6x V + A 02-07-2013 +
pSB6A1-AraC-lacZ (1:5 dilution of) 38.5 2 10* 1x - 02-07-2013 + (BackUP)
pSB6A1-AraC-lacZ -11 5 1* 6x V + A (Nucleotide Removal) 03-07-2013 +
Fig.10.7 gel of fragments (amplified by PCR) (loaded 20 µL)
l1: F1a, l2: F1a, l3: F1b (digested (D) & purified (P)), l4: F1b (D+P), l5: F2(D+P), l6: F2(D+P), l7: F2, l8: BB(D+P), l9: BB, l10: BB(D+P), l11: 2log
lane 2, 3, 4, 5 had no band

DelH F1a (40 ng/µl) was the fragment we used for the first ligation. It is not visible, thus, there was not enough little DNA. Since the screening primer binds to this part of DelH, we could not identify any positive clone.

=> The negative samples (with too little DNA concentration) are discarded.


Amplification of DelH F1a

PCR Conditions F1a.W10.C

Reagent DelH F1a
Template 1 µl DelH F1a Fragment 03-07-2013
Primer fw 10 µM 2.5 µl DelH_Pac_fw
Primer rev 10 µM 2.5 µl DelH_EcoRI_rev
Phusion Flash Ready Mix 25 µl
ddH2O 19 µl
Cycles-PCR Temperature [°C] Time [s]
1 98 10
30 98 1
66 5
72 120
1 72 7 min
1 4 inf

Result

Fragment was gel extracted.

Amplification of DelH F1b

PCR Conditions F1b.W10.D

Reagent DelH F1b DelH F1b
Template 1 µl D. acidovorans 1 µl DelH F1b Fragment (19 ng/µl)
Primer fw 10 µM 2.5 µl DelH_EcoRI_fw 2.5 µl DelH_EcoRI_fw
Primer rev 10 µM 2.5 µl DelH_f1_SalI_rev 2.5 µl DelH_f1_SalI_rev
Phusion Ready Mix 25 µl 25 µl
ddH2O 19 µl 19 µl
Cycles Temperature [°C] Time [s]
1 98 5
30 98 1
68 5
72 2:15 min
1 72 7 min
1 4 inf

Result

Expected band: 5 Kb

Fig.10.9 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

No bands visible.

=> Fragment F1b could not be amplified from neither genomic DNA nor PCR fragment. Amplification will be repeated using DMSO in the mix.


PCR Conditions F1b.W10.E

Reagent DelH F1b DelH F1b DelH F1b DelH F1b
Template 1 µl D. acidovorans glycerol stock 1 µl Fragment (19 ng/µl) 1 µl D. acidovorans glycerol stock 1 µl PCR Fragment (19 ng/µl)
Primer fw 10 µM 1 µl DelH_EcoRI_fw 1 µl DelH_EcoRI_fw 1 µl DelH_EcoRI_fw 1 µl DelH_EcoRI_fw
Primer rev 10 µM 1 µl DelH_f1_SalI_rev 1 µl DelH_f1_SalI_rev 1 µl DelH_f1_SalI_rev 1 µl DelH_f1_SalI_rev
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl
ddH2O 7 µl 7 µl 6 µl 6 µl
DMSO - - 1 µl 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
68 (touchdown -0.5°C) 5
72 2:15 min
18 98 1
68 5
72 2:15 min
1 72 7 min
1 4 inf
  • Using hot start 98°C

Result

Expected band: 5 Kb

Fig.10.10 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

Only PCR of fragment 19 ng/µl + DMSO was successful. Maybe there is another pale band at the second PCR of DelH F1b.

=> Repeat both PCRs.


Result

PCR using conditions F1b.W10.E was repeated.
Expected band: 5 Kb,Entire PCR mix was loaded.

Fig.10.12 gel of amplified fragments (loaded 20 µL)
l1/2: entire F1b, l3/4: older PCR of F1b, l5: 2log ladder, l6/7 BB
excised band of lane 6/7
Fig.10.11 gel of amplified fragments (loaded 20 µL)
l1/2: entire F1b, l3/4: older PCR of F1b, l5: 2log ladder, l6/7 BB
excised band of lane 6/7

PCR did not result in any fragments. Interestingly, both older PRCs resulted in a fragment at ~5 Kb and other lines, but the fragment from the genomic DNA is slightly larger.

=> Both fragments were cut and gel isolated, check correct length of F1b!
=> Perform PCR from the PCR fragment obtained from the genomic DNA.


Amplification of DelH F2

PCR Conditions F2.W10.C

  • 2x 50 µl
Reagent DelH F2
Template 1 µl Fragment "BackUP"
Primer fw 10 µM 2.5 µl DelH_f2_SalI_fw
Primer rev 10 µM 2.5 µl DelH_f2_KpnI_rev
Phusion Flash Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66 decrease by 0.5 5
72 2:30 min
18 98 1
66 5
72 2:30 min
1 72 7 min
1 4 inf

Result

Expected band: 8 Kb

Fig.10.9 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

Fragment 2 was successfully amplified.

=> It was cut and gel purified.


Preparation of new Template DNA

  • D. acidovorans glycerol stock was streaked onto LB plates and incubated ON at 37°C.
  • D. acidovorans did not grow, since LB media is not appropriate for them. Prepare new ACM media and plates and streak D. acidovorans.


Amplification of Backbone

PCR Conditions BB.W10.D

  • 2x 50 µl
Reagent Backbone
Template 1 µl Fragment "BackUP"
Primer fw 10 µM 2.5 µl AraCbb_KpnI_fw
Primer rev 10 µM 2.5 µl AraCbbPacI_rev2
Phusion Flash Ready Mix 25 µl
ddH2O 19 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66 decrease by 0.5 5
72 2:30 min
18 98 1
66 5
72 2:30 min
1 72 7 min
1 4 inf

Result

Expected band: 7.3 Kb

Fig.10.13 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

No band visible.

=> Repeat using DMSO.


PCR Conditions BB.W10.E

Reagent Backbone Backbone Backbone
Template 1 µl of BackUP 1 µl V+A 2,2 ng/µl template 1 µl V+A 2,2 ng/µl template
Primer fw 10 µM 1 µl AraCbb_KpnI_fw 1 µl AraCbb_KpnI_fw 1 µl AraCbb_KpnI_fw
Primer rev 10 µM 1 µl AraCbbPacI_rev2 1 µl AraCbbPacI_rev2 1 µl AraCbbPacI_rev2
Phusion Flash Ready Mix 10 µl 10 µl 10 µl
ddH2O 7 µl 7 µl 6 µl
DMSO - - 1 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66 decrease by 0.5 5
72 2:30 min
18 98 1
66 5
72 2:30 min
1 72 7 min
1 4 inf
  • Using hot start (in new cycler)

Result

Expected band: 7.3 Kb

Fig.10.10 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified

Gel does not show the expected fragment.

=> Next PCR for the Backbone should be done with longer elongation time and variations: think about using the a new colony of plate.


PCR Conditions BB.W10.F

Reagent Backbone Backbone Backbone Backbone
Template 1 µl Fragment 24 ng/µl 1 µl of Fragment pSB6A1 gel-purified 3-6 89 ng/µl 1 µl of digested Fragment 3-7 -11 ng/µl 1 µl of purified Fragment Isoprop 2.2 ng/µl
Primer fw 10 µM 1 µl DelH_f2_SalI_fw 1 µl AraCbb_KpnI_fw 1 µl DelH_f2_SalI_fw 1 µl AraCbb_KpnI_fw 1
Primer rev 10 µM 1 µl DelH_f2_KpnI_rev 1 µl AraCbbPacI_rev2 1 µl DelH_f2_KpnI_rev 1 µl AraCbbPacI_rev2
Phusion Flash Ready Mix 10 µl 10 µl 10 µl 10 µl
ddH2O 7 µl 7 µl 7 µl 7 µl
Cycles Temperature [°C] Time [s]
1 98 5
12 98 1
66 decrease by 0.5 5
72 180
18 98 1
66 5
72 180
1 72 7 min
1 4 inf

Result

Expected band: 7.3 Kb,Entire PCR mix loaded (pSB6A1-AraC-lacZ)

Fig.10.12 gel of amplified fragments (loaded 20 µL)
l1/2: entire F1b, l3/4: older PCR of F1b, l5: 2log ladder, l6/7 BB
excised band of lane 6/7
Fig.10.11 gel of amplified fragments (loaded 20 µL)
l1/2: entire F1b, l3/4: older PCR of F1b, l5: 2log ladder, l6/7 BB
excised band of lane 6/7

Only PCRs from Fragment 24 ng/µl and Fragment 3-6 produced a nice fragment.

=> Bands were cut and gel isolated.
=> PCR amplification from restriction digested fragment did not work! This is unexpected, because actually, it should. Maybe exclude the previous restriction digested fragments "V+A" from ligation.


Preparation of new Template DNA

  • psB6A1-LacZ-AraC: 2 ml LB Amp were inocculated from the plate and incubated ON at 28°C (all other shakers occupied).
  • Mini prep was performed.
  • 1 µl mini prep DNA was electroporated into E.coli DH10ß according to Electroporation of E. coli DH10β


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