Team:Heidelberg/Templates/Del week11 OP

From 2013.igem.org

Contents

09-07-2013

Amplification I from FS_12 to FS_13; 2.7 kb

Analyticals gel older fragments + PCR for amplification of del fragments DelL (08.07), and DelOP (09.07); lane1=Marker, lanes2-7= DelAF (11.4kbp), BB psB4K5 (4.4kbp), DelOP (2.7kbp), DelG (6.4kbp), DelL (1.4kbp), DelAE (5.3kbp), DelEG (11.0kbp), lanes8-10=DelL (1.4kbp), lanes11-13:DelOP (2.5kbp); run at 100 V, 0.8 % gel (TAE)
Analyticals lane1=Marker, lanes2-7= DelAF (11.4kbp), BB psB4K5 (4.4kbp), DelOP (2.7kbp), DelG (6.4kbp), DelL (1.4kbp), DelAE (5.3kbp), DelEG (11.0kbp) + PCR for amplification of del fragments DelL (08.07), and DelOP (09.07), cut
Reaction
what µl
D. acidovorans DSM-39 1
FS_12: (1/10) 1
FS_13: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 40
1 72 7 min
1 12 inf

Results:

  • Amplification of DelOP did not work
  • PCR was repeated, Annealing was carried out as touchdown starting from 68°C

Amplification II from FS_12 to FS_13; 2.7 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_12: (1/10) 1
FS_13short: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 6
8 98 1
68 ↓ 0.5 5
72 50
24 98 1
72 5
72 50
1 72 11min
1 4 inf

Results:

  • Amplification of DelOP did not work
  • PCR will be repeated with the newly ordered short version of primer FS_13, testing different DMSO settings

10-07-2013

Amplification from FS_12 to FS_13(s); 2.7 kb

PCR for amplification of DelOP, DelFG and DelG; +=with DMSO, -=without DMSO, L=both primers are the long primers, S= one Primer is one of the short primers; lane1=Marker, lane2=delOP(04.07,L+), lane3=DelOP(10.07,L+), lane4=DelOP(10.07,L-), lane5=DelOP(10.07,S+), lane6=DelOP(10.07,S-), lane7=DelFG(10.07,+), lane8=DelFG(10.07,-), lane9=DelOP(09.07,S+), lane10=DelG(09.07,S+); expected amplicon sizes: DelOP=2.5kbp, Del DelFG=ca 5kbp, DelG=6.4kbp; run at 100 V, 0.8 % gel (TAE)
Gel after excision
Reaction of DelO-P (2.6 kb)

4 reactions with different conditions: with/without DMSO, short/long Primer FS13

what µl
D. acidovorans DSM-39 1
FS_12: (1/10) 1
FS_13(long or short): (1/10) 1
Phusion Master Mix 10
DMSO 1/-
dd H2O 6/7
Conditions of Del O-P
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 40
1 72 420
1 12 inf

Results:

  • None of the Amplification neither with primer FS_13 nor with FS_13short worked out, nevertheless did addition of DMSO change the amplification result
  • PCR will be carried out as 2-step in another more precise cycler
  • PCR will be set up with as touchdown PCR, annealing starting at 68°C
  • Furthermore a modified version of the forward primer will be order. This primer (FS_22) includes a recently in the Del Cluster predicted promotor as well as a likewise predicted ribosome binding site
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