Team:Heidelberg/Templates/Del week16 pSB4K5

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Contents

15-08-2013

Amplification from FS_01 to FS_16; 4.2 kb

Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Amplification of DelFG and Backbone pSB4K5; run at 100 V, 0.8 % gel (TAE)(14.08)
Reaction

(2x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad T100
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf

Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 15-08-2013) with DpnI

Incubation at 37°C for about 6 hours

what µl
FS_16 to FS_01 (15-08-2013) 30
DpnI 2
CutSmart Buffer 4
dd H2O 4

Results:

  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

16-08-2013

Concentration measurement

The concentration of the gel purified, and DpnI digested fragment was measured using a NanoDrop Instrument.

Fragment Primer Date PCR Concentration
pSB4K5 DpnI digested FS01-FS16 15-08-2013 30 ng/µl

Amplification from FS_01 to FS_16; 4.2 kb

Amplification of pSB4K5 (16-08); run at 100 V, 0.8 % gel (TAE)
Amplification of pSB4K5; cut (16-08); run at 100 V, 0.8 % gel (TAE)
Reaction

(3x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad T100
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf


Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

17-08-2013

Amplification from FS_01 to FS_16; 4.2 kb

Reaction

(3x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf


Results:

  • Amplification worked very well, we had bright bands on the right height.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 17-08-2013) with DpnI

Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP (18-08); run at 100 V, 0.8 % gel (TAE)
Restriction digest of pSB4K5 (17-08) with DpnI (17-08) and amplification of DelOP, cut (18-08); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 6 hours

what µl
FS_16 to FS_01 (17-08-2013) 30
DpnI 2
CutSmart Buffer 4
dd H2O 4

Results:

  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

18-08-2013

Amplification from FS_01 to FS_16; 4.2 kb

Reaction

(3x50µl)

what µl
Template pSB4K5 1
FS_01: (1/10) 2.5
FS_16: (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 16.5


Conditions
Biorad MyCycler*
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
62 5
72 1:30 min
1 72 5 min
1 8 inf

Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 18-08-2013) with DpnI

Incubation at 37°C for about 6 hours

what µl
FS_16 to FS_01 (18-08-2013) 30
DpnI 2
CutSmart Buffer 4
dd H2O 4

Results:

  • Digested fragment was run on a gel.
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
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