Team:Heidelberg/Templates/DelH week2

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Contents

06-05 - 12-05-13

Amplification of BioBricks

Miniprep of BioBricks

  • Miniprep was performed using Qiagen kit and eluted in 35 µl ddH2O.
  • From 500 µl of liquid culture, a glycerol stock was prepared as described in Preparing glycerol stocks.

Result

  • DNA concentration was determined using nanovue.
Sample Concentration [ng/µl]
pSB4K5 21
pSB6A1 47
lacZ 45
pSB1C3 23


Generation of Plasmid Backbones

Restriction Digest of BioBrick pSB6A1

Component Amount [µl]
PSB6A1 DNA 32.5
BSA (10x)5
NEB2 buffer (10x) 5
Enzymes (EcoRI-HF and PstI) 1.5 each
ddH2O 4.5

Result

Fig. 2.1: Restriction results of 06-05. l1:2log, l2: P1 (pSB4K5),l3:2log, l4-5:amplified DelH fragment 1 , l6-7:amplified DelH fragment 2,l8:2log, l9: P3 (pSB1C3)


The fragments of plasmids pSB4K5 and pSB1C3 show the expected pattern.

=> BioBrick backbone was cut and gel isolated.


Generation of LacZ Fragment

Restriction Digest of BioBrick pSB1AK3 Conditions A

Component Amount [µl]
pSB1AK3 DNA 32.5
BSA (10x)5
NEB3 buffer (10x) 5
Enzymes (XbaI and PstI) 1.5 each
ddH2O 4.5
  • Incubation for 1 h at 37°C

Result

  • The restriction mix was loaded onto a 1% agarose gel using 6 µl gene ruler marker.
Fig. 2.1: Restriction results of 06-05. l1:2log, l2: P1 (pSB4K5),l3:2log, l4-5:amplified F1 for Backbone (pSB6A1), l6-7:amplified fragment 2 for Backbone (lacZ),l8:2log, l9: P3 (pSB13)

The fragments show the expected pattern.

=> LacZ insert were cut and gel isolated.


Restriction Digest of BioBrick K173004(lacZ) Conditions B

Component Amount [µl]
pSB1AK3 DNA 8.3
BSA (10x) 5
NEB4 buffer (10x) 5
Enzymes (XbaI & SalI-HF) 1.5 each
ddH2O 28.7
  • Purification was performed with Qiagen Nucleotide removal Kit and eluted in 38.5 µl ddH2O

Result

2.5 µl were measured with the Nanovue. The measurement proved the failure of miniprep.

Restriction Digest of BioBrick K173004(lacZ) Conditions C

Component Amount [µl]
pSB1AK3 DNA 32.5
BSA (10x) 5
NEB 3 buffer (10x) 5
Enzyme (PstI) 1.5
ddH2O -

Result

Fig.2.2 Gel of cut lacZ (loaded 20 µL)
, l1-2: lacz & pSB1AT,l3:2log ladder
=> lacZ was cut out


=>The band was too high on gel and discarded, because we cannot be sure if it really is lacZ or the backbone of the lacZ plasmid.



Generation of AraC Fragment

Amplification of BioBricks containing I13453 and K206000

  • The transformation was performed as described on 03-05 and incubated ON at 37°.


Amplification of DelH F1

PCR Conditions F1.W2.A

Reagent DelH F1 DelH F1
Expected length [Kb] 10 10
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans DNA
Primer 100 µM fw 0.5 µl DelH_f1_PacI_fw 0.5 µl DelH_f1_PacI_fw
Primer 100 µM rev 0.5 µl DelH_f1_SalI_rev 0.5 µl DelH_f1_SalI_rev
Phusion Master Mix (2x) 25 µl 25 µl
ddH2O 23 µl 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
16 98 1
66(↓0.5°C) 5
72 3:40 min
14 98 1
62 5
72 3:40 min
1 72 5 min
1 4 inf

Result

Expected band: 10 Kb

Fig.2.3 Gel of amplified DelH-fragment 1 & fragment 2 (loaded 20 µL)
l1:2log ladder, l2-3:DelH-F1, l1:2log ladder, l5-6:DelH-F2
no specific band at 10 Kb

Different unspecific bands were observed.

=> Further optimize PCR conditions.


PCR Conditions F1.W2.B

Reagent DelH-fragment1 DelH-fragment1
Expected length [Kb] 10 10
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans DNA
Primer 100 µM fw 0.5 µl DelH_f1_PacI_fw 0.5 µl DelH_f1_PacI_fw
Primer 100 µM rev 0.5 µl DelH_f1_SalI_rev 0.5 µl DelH_f1_SalI_rev
Phusion Master Mix (2x) 25 µl 25 µl
ddH2O 23 µl 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1
72 2:45 min
1 72 5 min
1 4 inf

Result

Fig.2.4 Gel of amplified DelH-fragment 1 (loaded 20 µL)
l1:2log ladder, l2:DelH-F1 amplified from DNA, l3:DelH-F1 amplified from cells
no specific band at 10 Kb

PCRs showed only unspecific bands.

=> Further optimize PCR conditions.


PCR Conditions F1.W2.C

Reagent DelH F1 DelH F1 DelH F1 DelH F1
Expected length [Kb] 10 10 10 10
Template 1 µl D. acidovorans normal 1 µl D. acidovorans normal 1 µl D. acidovorans 1:25 1 µl D. acidovorans 1:25
DelH_f1_PacI_fw 0.5 µl normal 0.5 µl 1:10 0.5 µl normal 0.5 µl 1:10
DelH_f1_SalI_rev 0.5 µl normal 0.5 µl 1:10 0.5 µl normal 0.5 µl 1:10
Phusion Master Mix (2x) 25 µl 25 µl 25 µl 25 µl
DMSO - - 2.5 µl 2.5 µl
ddH2O 23 µl 23 µl 20.5 µl 20.5 µl
Cycles Temperature [°C] Time [s]
1 98 10
16 98 1
62 5
72 2:45 min
1 72 5 min
1 4 inf

Result

Fig.2.5 Gel of DelH-fragment 1 (loaded 20 µL)
l1:2log ladder, l2-9: F1 with different conditions => l9 shows the best match

PCR with DMSO and 1:10 diluted primer looked best.

=> Gel extraction performed.


Amplification of DelH F2

PCR Conditions F2.W2.A

Reagent DelH F2 DelH F2
Expected length [Kb] 8 8
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans DNA
Primer 100 µM fw 0.5 µl DelH_f2_SalI_fw 0.5 µl DelH_f2_SalI_fw
Primer 100 µM rev 0.5 µl DelH_f2_KpnI_rev 0.5 µl DelH_f2_KpnI_rev
Phusion Master Mix (2x) 25 µl 25 µl
ddH2O 23 µl 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
16 98 1
66(↓0.5°C) 5
72 3:40 min
14 98 1 s
62 5 s
72 3:40 min
1 72 5 min
1 4 inf

Result

Expected band: 8 Kb

Fig.2.3 Gel of amplified DelH-fragment 1 & fragment 2 (loaded 20 µL)
l1:2log ladder, l2-3:DelH-F1, l4:2log ladder, l5-6:DelH-F2
no specific band at 8 Kb

Different unspecific bands were observed.

=> Further optimize PCR conditions.


PCR Conditions F2.W2.B

Reagent DelH F2 DelH F2
Expected length [Kb] 8 8
Template 1 µl D. acidovorans glycerol stock 1 µl D. acidovorans DNA
Primer 100 µM fw 0.5 µl DelH_f2_SalI_fw 0.5 µl DelH_f2_SalI_fw
Primer 100 µM rev 0.5 µl DelH_f2_KpnI_rev 0.5 µl DelH_f2_KpnI_rev
Phusion Master Mix (2x) 25 µl 25 µl
ddH2O 23 µl 23 µl
Cycles Temperature [°C] Time [s]
1 98 10
30 98 1 s
72 2:45 min
1 72 5 min
1 4 inf

Results

Expected band: 8 Kb

Fig.2.6 Gel of amplified DelH-fragment 1 (loaded 20 µL)
l1:DelH-F1 amplified from DNA, l2:DelH-F1 amplified from cells, l3:2log ladder
no specific band at 8 Kb

Gel shows the expected specific band at 8 Kb.

=> DelH F2 was thus successfully amplified from the glycerol stock.



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