Team:Heidelberg/Templates/DelH week20

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<div class="tright" style="clear:none">[[File:Heidelberg_20130915 2log 6xFS77 6xHM20 58.png|200px|thumb|right|'''Fig.20.20.''' Restriction digested BB pSB6A1 with new primer and primer HM17 at an annealing temperature of 58°C (loaded 20 µL of PCR) <br> ''l1:''2 log ladder,''l2-7:''BB amplified HM17 and FS77,''l8-13:''BB amplified HM17 and HM20 - specific band at 4 Kb]]</div>
<div class="tright" style="clear:none">[[File:Heidelberg_20130915 2log 6xFS77 6xHM20 58.png|200px|thumb|right|'''Fig.20.20.''' Restriction digested BB pSB6A1 with new primer and primer HM17 at an annealing temperature of 58°C (loaded 20 µL of PCR) <br> ''l1:''2 log ladder,''l2-7:''BB amplified HM17 and FS77,''l8-13:''BB amplified HM17 and HM20 - specific band at 4 Kb]]</div>
   
   
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Gel shows expected bands.
Gel shows expected bands.
:=> Fragments were gel extracted.
:=> Fragments were gel extracted.
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===Generation of DelH Plasmid pHM04 15-09===
===Generation of DelH Plasmid pHM04 15-09===
====Gibson Assembly====
====Gibson Assembly====

Latest revision as of 21:22, 25 October 2013

Contents

09-09 - 15-09-13

Characterization of DelH Plasmid pHM04 30-08 Clones 4, 7, 15

Colony-PCR Conditions CP.W20.A

Because the sequenced DelH-colonies 4, 7, 15 had different kinds of mutations (Deletion of one basepair but also an insertion of a whole sequence part => results are shwon in week 19) we made a new screening PCR of 30 new picked colonies named 31-60 of the plate 2 of the electroporated cells realized in week 19.
There were 30 colonies screened.

Template 30x 1 PC of S-A
Expected length [bp] 663
Named 31-60
Primer fw 10 µM 30x 2 µl VF2
Primer rev 10 µM 30x 2 µl DN07
iTaq Polymeras (2x) 30x 10 µl
ddH2O 30x 6 µl
Cycles Temperature [°C] Time [s]
1 95 120
12 95 60
68 (touchdown -0.5°C) 30
72 45
18 95 60
65 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.20.2 Colony PCR of Gibson assembled DelH-BB without mRFP (loaded 10 µL of PCR)
l1:50 bp ladder, l2-15:colonies 47-60, l16: ddH2O
l6, l13:show the specific band at 663 bp
Fig.20.1 Colony PCR of Gibson assembled DelH-BB without mRFP (loaded 10 µL of PCR)
l1:50 bp ladder, l2-17:colonies 31-46
l4, l10,l11,l17:show the specific band at 663 bp


Colonies 33, 39, 40, 46, 51, 58 showed band.

=> Send in for sequencing after isopropanol ethanol purification.


Sequencing

The colonies 33, 39, 40, 46, 51, 58 were sent in MWG for sequencing. There for we prepared 15 µl of the plasmid (midiprep) with a concentration of 50-100 ng/µl and add 2 µl DN07 primer (10µM).

Result

Colony Alignment File Conclusion
H33 sequencing insufficient
H39 sequencing insufficient
H40 sequencing insufficient
H46 sequencing insufficient
H51v sequencing insufficient
H58 File:Heidelberg Sequencing Result pHM04 DN07 colony H58 DN07.clustal.txt insertion in the primer region of DelH-backbone (pHM04)


Colony-PCR Conditions CP.W20.B

New picked olonies 2x 95 well plate

Template 95x 1 µl of colony
Expected length [bp] 663
Named I 1A - I 12H , II 1A - II 12H
Primer fw 10 µM 105x 2 µl VF2
Primer rev 10 µM 105x 2 µl DN07
iTaq Polymerase (2x) 105x 10 µl
ddH2O 105x 5 µl
Cycles Temperature DelH [°C] Time [s]
1 95 120
12 95 30
68 (touchdown -0.5°C) 30
72 45
18 95 30
65 30
72 45
1 12 inf

Result

Expected band: 663 bp

Fig.20.10 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 9H-12H
Fig.20.9 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l2-l26:colonies 6G-9G
Fig.20.8 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l24:colonies 3H-6F
Fig.20.7 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate II (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 1A-3G
Fig.20.6 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 10A-12H
b
Fig.20.5 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 7C-9H
Fig.20.4 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I (loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 4B-7B
Fig.20.3 Colony PCR of Gibson assembled DelH-BB without mRFP 96 well plate I(loaded 10 µL of PCR)
l1:50 bp ladder, l2-l26:colonies 1A-4A

Colonies collected in table below show a definit result.

=> Add medium and perform a mediprep the next day for send in for sequencing:
Colony of plate I Colony of plate II
1F 2D
1G 2C
2A 3G
2H 4A
3B 4E
4C 7C
4E 7G
4F 8B
4H 10E
5E 10H
5F 11A
5G
(5H)
6B
6C
6D
7H
8B
8D
8F
8G
9B
9C
9F
10B
10C
10G
11F
12G
=> The next step is a test restriction digest with PvuI


Test Restriction Digest

We performed a test digest with PvuI-HF of the screened samples which showed a postive band at 663 bp in the screening PCR. Therefore we prepared a Master Mix with:

Enzyme CutSmartBuffer [µl] ddH2O [µl] Total amount [µl]
45x 0.5 µl = 22.5 µl 45x 2 µl = 90 µl 45x 16.5 µl = 742.5 µl 45x 19 µl = 855 µl
  • Incubation time: 1:20 h at 37 °C

Result

Expected bands: 11.5 Kb, 8.5 Kb and 2.6 Kb.

Fig.20.14. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:4E,l3: 7C,l4: 7G,l5: 8A,l6: 8B,l7:10E,l8: 10H,l9:11A
Fig.20.13. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:plate I 10A,l3: 10B,l4: 10C,l5: 10D,l6: 10G,l7: 11F,l8: 12G,l9: plateII 2B,l10: 2D,l11: 2C,l12: 3G,l13: 4A
Fig.20.12. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:6B,l3: 6C,l4: 6D,l5: 7H,l6: 8B,l7:8D,l8: 8F,l9:8G,l10: 9B,l11: 9C,l12:9F,l13: 10C
Fig.20.11. Test digest of pHM04 from different colonies with PvuI (loaded 20 µL of PCR)
l1:2log,l2:1F,l3: 1G,l4: 2A,l5: 3B,l6: 4C,l7: 4E,l8: 4F,l9: 4H,l10: 5E,l11: 5F,l12: 5G,l13: 5H

Some colonies showed the expected bands (see table below).

=> These are sent in for sequencing after a miniprep.

The table below shows the result of the restriction digest of different colonies containing the pHM04 plasmid. The restriction digest was positive if the expected bands were pesent.

Colony of plate I Show expected bands Colony of plate II Show expected bands
1F - 2D -
1G - 2C -
2A - 3G +
2H - 4A -
3B - 4E +
4C - 7C -
4E + 7G +
4F - 8B +
4H + 10E -
5E - 10H -
5F - 11A -
5G - - -
(5H) - - -
6B + - -
6C + - -
6D + - -
7H + - -
8B - - -
8D - - -
8F - - -
8G + - -
9B - - -
9C - - -
9F - - -
10B + - -
10C - - -
10G + - -
11F + - -
12G - - -
Colony Concentration ng/µl
I 4E 1597
I 4H 495
I 6B 255
I 6C 2445
I 6D 2101
I 7H 1539
I 8G 1589
I 10B 1359
I 10G 1863
I 11F 1886
II 3G 564
II 4E 2107
II 7G 1913
II 8B 955


Sequencing

Colony of plate I Concentration ng/µl Amount for sequencing [µl] ddH2O [µl] Primer (DN07 [10 µM]) added [µl]
I 4E 1597 1 14 2
I 4H 495 1.5 13.5 2
I 6B 255 5 10 2
I 6C 2445 0.5 14.5 2
I 6D 2101 0.5 14.5 2
I 7H 1539 1 14 2
I 8G 1589 1 14 2

Result

Colony Sequencing Notes Alignment File
I 4E - (Deletion of G in coding sequence) File:Heidelberg Sequencing Result pHM04 DN07 colony I4E.clustal.txt
I 4H - (Deletion of G in coding sequence) File:Heidelberg Sequencing Result pHM04 DN07 colony I4H.clustal.txt
I 6B + ? a deletion in the RBS File:Heidelberg Sequencing Result pHM04 DN07 colony I6B.clustal.txt
File:Heidelberg Sequencing Result pHM04 VF2 colony 6B VF2 VF 2.clustal.txt
I 6C - (9 deletion) File:Heidelberg Sequencing Result pHM04 DN07 colony I6C.clustal.txt
I 6D - Deletion File:Heidelberg Sequencing Result pHM04 DN07 colony I6D.clustal.txt
I 7H - Deletion File:Heidelberg Sequencing Result pHM04 DN07 colony I7H.clustal.txt
I 8G - Deletion File:Heidelberg Sequencing Result pHM04 DN07 colony I8G.clustal.txt
I 8B - Deletion of G in coding sequence discarded
I 10B - Deletion of G in coding sequence discarded
I 10G - Insertion of G in coding sequence discarded
I 11F - Deletion of G in coding sequence discarded
II 3G - G Deletion of the ATG (start-codon) and 3 bp later deletion of C discarded
II 4E - Deletion of G in coding sequence discarded
II 7G - G Deletion of the ATG (start-codon) and 3 bp later deletion of C discarded

Because the colony 6B is possibly positive, the next steps are:

1. SDS-PAGE
2. Sequencing over gibson-assembled parts
3. Triple electroporation with the plasmig of Del-rest and MalonylCoA (see lab book Delftibactin)



Amplification of Backbone pSB6A1

PCR Conditions BB.W20.A

Reagent BB pSB6A1 BB pSB6A1
Expected length [Kb] 4.4 4.4
Template 0.5 µl pSB6A1 30-8 0.5 µl pSB6A1 30-8
Primer 10 µM fw 2 µl HM_17 2 µl HM_17
Primer 10 µM rev 2 µl FS_77 2 µl FS_77
Phusion Flash Master Mix (2x) 10 µl 10 µl
DMSO 1 µl 1 µl
ddH2O 4.5 µl 4.5 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
68↓ 5
72 3:00 min
18 98 1
66 5
72 3:00 min
1 72 5:00 min
1 4 inf
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
72 3:00 min
1 72 5:00 min
1 4 inf

Result

Expected band: 4.4 Kb

Fig.20.15. Amplification of BB pSB6A1 with new primer and primer HM17 (loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and FS77 (68td),l3:BB amplified HM17 and HM20 (68td),l4: BB amplified HM17 and FS77 (2step),l5: BB amplified HM17 and HM20 (2step) - no yield

Very weak amplification, Primer-Dimers/Oligomers, carry over of Backbone pSB6A1 (including mRFP and therefore creating a double band, since template is 700bp longer than expected PCR product).

=> Repeat PCR with less stringent conditions to increase yield until unexpected bands appear or yield is high enough and reduce amount of template DNA (use 0.5 µL of a 1:10 delution)
=>Furthermore, elongation time was reduced, as template is only about 4.4 Kb long instead of the putative 7 Kb.


PCR Conditions BB.W20.B

Reagent BB pSB6A1 BB pSB6A1
Expected length [Kb] 4.4 4.4
Template 0.5 µl pSB6A1 (3 ng/µL) 0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw 2 µl HM_17 2 µl HM_17
Primer 10 µM rev 2 µl FS_77 2 µl FS_77
Phusion Flash Master Mix (2x) 10 µl 10 µl
DMSO 1 µl 1 µl
ddH2O 4.5 µl 4.5 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
66↓ 5
72 80
18 98 1
64 5
72 80
1 72 5:00 min
1 4 inf
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 80
1 72 5:00 min
1 4 inf

Result

Expected band: 4.4 Kb

Fig.20.16. Amplification of BB pSB6A1 with new primer and primer HM17 with different PCR conditions(loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and HM20 (66°C),l3:BB amplified HM17 and FS77 (66°C),l4:BB amplified HM17 and HM20 (58°C), l5:BB amplified HM17 and FS77 (58°C) - l4-5 show the expected band at 4.4 KB

Gel shows best amplification of fragment using annealing temperature at 58°C.

=> Repetition of PCR at a constant annealing temperature of 58°C.


PCR Conditions BB.W20.C

Performed 6x PCR with FS77 & 6x PCR with HM20, so we have enough yield for the Gibson assembly.

Reagent BB pSB6A1 BB pSB6A1
Expected length [Kb] 4.4 4.4
Template 0.5 µl pSB6A1 (3 ng/µL) 0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw 2 µl HM_17 2 µl HM_17
Primer 10 µM rev 2 µl FS_77 2 µl FS_77
Phusion Flash Master Mix (2x) 10 µl 10 µl
DMSO 1 µl 1 µl
ddH2O 4.5 µl 4.5 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 80
1 72 5:00 min
1 4 inf

Result

Expected band: 4.4 Kb

Fig.20.17. Amplification of BB pSB6A1 with new primer and primer HM17 with a constant annealing temperature at 58°C(loaded 20 µL of PCR)
l1:2 log ladder,l2-7:BB amplified HM17 and FS77,l8-13:BB amplified HM17 and HM20 - no bands visible

Gel does not show the expected bands.

=> What happened? Why is it not reproducible? Run a PCR with other conditions.


PCR Conditions BB.W20.D

Reagent BB pSB6A1 BB pSB6A1
Expected length [Kb] 4.4 4.4
Template 1 µl pSB6A1 (3 ng/µL) 1 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw 2 µl HM_17 2 µl HM_17
Primer 10 µM rev 2 µl FS_77 2 µl FS_77
Phusion Flash Master Mix (2x) 10 µl 10 µl
ddH2O 5.0 µl 5.0 µl
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
60↓ 5
72 80
18 98 1
68 5
72 80
1 72 5:00 min
1 4 inf

Result

Expected band: 4.4. Kb

Fig.20.18. Amplification of BB pSB6A1 with new primer and primer HM17 with a td PCR (60°C) and a constant annealing temperature of 68°C(loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and FS77,l3:BB amplified HM17 and HM20 - no yield

Gel shows again no amplification of backbone.

=> Further optimize PCR conditions.


PCR Conditions BB.W20.E

Reagent BB pSB6A1 BB pSB6A1
Expected length [Kb] 4.4 4.4
Template 0.5 µl pSB6A1 (3 ng/µL) 0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw 1 µl HM_17 1 µl HM_17
Primer 10 µM rev 1 µl FS_77 1 µl FS_77
Phusion Flash Master Mix (2x) 10 µl 10 µl
DMSO 1 1
ddH2O 6.5 µl 6.5 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
58 5
72 80
1 72 5:00 min
1 4 inf
Cycles Temperature [°C] Time [s]
1 98 10
12 98 1
66↓ 5
72 1:20 min
18 98 1
64 5
72 80
1 72 5:00 min
1 4 inf

Result

Expected band: 4.4 Kb

Fig.20.19. Amplification of BB pSB6A1 with new primer and primer HM17 (loaded 20 µL of PCR)
l1:2 log ladder,l2:BB amplified HM17 and HM20 (66td),l3:BB amplified HM17 and FS77 (66td),l4: BB amplified HM17 and HM20 (58°C),l5: BB amplified HM17 and FS77 (58°C) - l4-5 show specific band - was cut out

Gel shows amplification using new HM17.

=> Fragments were cut and gel extracted.


PCR Conditions BB.W20.F

Run 6x PCR with HM20 and 6x PCR with FS77 for higher yield

Reagent BB pSB6A1 BB pSB6A1
Expected length [Kb] 4.4 4.4
Template 0.5 µl pSB6A1 (3 ng/µL) 0.5 µl pSB6A1 (3 ng/µL)
Primer 10 µM fw 1 µl HM_17 1 µl HM_17
Primer 10 µM rev 1 µl FS_77 1 µl FS_77
Phusion Master Mix (2x) 10 µl 10 µl
DMSO 1 1
ddH2O 6.5 µl 6.5 µl
Cycles Temperature [°C] Time
1 98 10
30 98 1
58 5
72 80
1 72 5:00 min
1 4 inf

The PCR was direcly digested with DpnI afterwards.

Restriction Digest with DpnI

  • 10 of the 12 PCRs (5x FS & 5x HM) were cut with DpnI. There we used the exact PCR product as generated earlier.
  • Incubated at 37°C ON
  • The following table presents the amount for all reactions
Sample Buffer Enzyme ddH2O
10x 20 µl PCR 10x 2.5 µl CutSmart Buffer 2.5 µl DpnI 23 µl

Afterwards, a 0.8% gel was run for 1 h at 100 V and a gel-purification was performed with the Qiagen Gel Extraction Kit

Result

Expected band: 4.4 Kb

Fig.20.21. Restriction digested BB pSB6A1 with new primer and primer HM17 at an annealing temperature of 58°C (loaded 20 µL of PCR)
l1:2 log ladder,l2-7:BB amplified HM17 and FS77,l8-13:BB amplified HM17 and HM20 - specific band at 4 Kb
Fig.20.20. Restriction digested BB pSB6A1 with new primer and primer HM17 at an annealing temperature of 58°C (loaded 20 µL of PCR)
l1:2 log ladder,l2-7:BB amplified HM17 and FS77,l8-13:BB amplified HM17 and HM20 - specific band at 4 Kb

Gel shows expected bands.

=> Fragments were gel extracted.
Sample DpnI digested Concentration [ng/µl]
BB 16 (amplified with FS77) yes 6.2
BB 16 (amplified with HM20) yes 6.6


Generation of DelH Plasmid pHM04 15-09

Gibson Assembly

Fragment Concentration [ng/µl] Amount with BB16 (FS77) [µl] Amount with BB16 (HM20) [µl]
DN11-FS66 172.5 0.78 0.81
FS67-FS68 120.7 0.95 0.99
FS69-FS70 211.3 0.94 0.98
FS71-HM08 146 0.55 0.57
BB16 amplified with FS77 6.2 6.78 -
BB16 amplified with HM20 6.6 - 6.64
  • Incubate for 1h at 50°C

Electroporation

  • Afterwards, 5 µl of each Gibson assembly were taken out and 10 µl ddH2O was added
  • With 10 µl of the Gibson assembly, isopropanol purification was performed
  • 6 different electroporations were performed (see scheme below)
Electroporation name Inserted amount of Gibson Assembly Plated out on agar plates
HM20 1 1 µl (Gibson + ddH2O) * 10 µl * rest
HM20 14 14 µl (Gibson + ddH2O) * 10 µl * rest
HM20 20 20 µl (isopropanol purified) * 10 µl * rest
FS77 1 1 µl (Gibson + ddH2O) * 10 µl * rest
FS77 14 14 µl (Gibson + ddH2O) * 10 µl * rest
FS77 20 20 µl (isopropanol purified) * 10 µl * rest


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