Template:Heidelberg/notebook/pc/week33
week number 33
▼2015-08-10 PCR optimized
|
Ve |
Ve with DMSO |
Phusion |
|
5µl 5xbuffer |
5µl 5xbuffer |
5µl 5xbuffer |
|
2x1.25µl primer |
2x1.25µl primer |
2x1.25µl primer |
|
2.5µl 10mM dNTP |
2.5µl 10mM dNTP |
|
|
0.5 µl DNA |
0.5 µl DNA |
0.5 µl DNA |
|
0.25 Ve |
0.25 Ve |
12.5 MM |
|
14.25µl water |
13.25µl water |
9.5 µl water |
|
|
1µl DMSO |
|
Program:
95°C 2min
95°C 30sec
63-54°C 30sec
72°C 45 sec
72°C 5min
PCR purification with two of this samples gave to less DNA.
▼2015-08-11 Fragment check for Golden Gate
Fragment SH11 does not have the right side. New primers to build SH11 were ordered.
▼2015-08-11 Golden Gate
construct assembly, Golden Gate [Flo's recept?]
> ═════════════════════════════════════════════════
>
> • 15 µl raction mixture
> • 20 - 30 ng Backbone-Fragment
> • 1.5x des Insert-Fragments (in molar)
> • 1.5 µl NEBuffer 2.1
> • 1 µl NEB T4 Ligase (400 U / µl)
> • 0.75 µl BbsI (5 U / µl)
> • 1 mM ATP, 1 mM DTT (1.5 µl of 10 mM ATP/DTT stock)
>
> • Cycler program:
> ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
> cycle temp time purpose
> ────────────────────────────────────────────────────────
> 50x (or less) 37 °C 2:00 restriction reaction
> – 16 °C 5:00 ligation reaction
> ────────────────────────────────────────────────────────
> 1x 50 °C 5:00 ligase inactivation
> ────────────────────────────────────────────────────────
> 1x 80 °C 5:00 restr. enzyme inactivation
> ────────────────────────────────────────────────────────
> 1x 12 °C inf hold
▼2015-08-12 E.coli Trafo
The Entryvector contains a BsaI restriction side in the Amp. resistance. That is why an additional ligation step was added.
1µl T4 DNA Ligase
20°C 20min
65°C 10min
With this product DH5alpha Transformation was performed following the usual protocol. Transformation was done with 0.5µl, 2µl and rest DNADurchführung
▼2015-08-13 Colony PCR
All Transformations worked well. Colony PCR was performed.
5µl OneTag MM
2x 0.5µl primer
4µl water
95°C 4min
95°C 30 sec
55°C 30 sec
68°C 2min
68°C 3min
Fragments with the side of 1094bp were expected.
Fragments with the side of 1112bp were expected.
▼2015-08-14 Miniprep, test digest, Trafo
With all 16 colonies Mini Prep and a test digest were performed.
PStI Hf
BsaI Hf
1µl DNA
2µl SmartCut
2x 0.5µl Enzyme
16.5 µl water
Expected Fragments:
935 bp, 2038bp, 4892bp
Expected Fragments:
951 bp, 2043bp, 4924bp
Sample SH09L1 and JD28L1 were used for yeast transformation. For this co-transformation the Entryvector g2 and either SH09L1 or JD28L1 were used.
500ng from each DNA
7.7 µl DMSO
Glycerol stocks were made from Entryvector g2, g5, SH09L1 and JD28L1
▼2015-08-15 Cloning ccdB in p415 via restriction enzymes
p415 digest:
2µl CutSmart
1µl PstI HF
1µl HindIII HF
1.9 µl p415 (1µg)
14.1 nuclease free water
ccdBg:
2µl CutSmart
1µl Psti HF
1µl HindIII HF
9.38 µl ccdBg
6.62 water
ccdBk:
2µl CutSmart
1µl Psti HF
1µl HindIII HF
2.7 µl ccdBk
13.3 µl water
At 37°C over night
PCR purification
Ligation
ccdBg:
2 µl 10x T4 DNA Buffer
1µl p415 (50ng= 0.0111pmol)
0,52 µl ccdB (25.8ng= 0.0557pmol)
1µl T4 Ligase
15.48 µl water
E. coli trafo with ccdB resistant E.coli ccdBg or ccdBk or efficient check
Efficient check with Transformation Efficiency Kit BBa_J04450 psB1C3
ccdBg or ccdBk:
5 µl DNA
10 µl KCM 5x
35 µl water
Rest see protocol “KCM Transformation E.coli
▼2015-08-15 yeast colony PCR
Colonies from PC SH09L1 and PC JD28L1 plates were taken, put in –His-Leu medium and a colony pcr was performed.
Colony PCR to check yeast transformation
Sample preparation:
Pick a small amount of cells with pipet tips and transfer in 5µl water
Add 20µl 20mM NaOH each
Heat samples at 99°C for 10min using PCR machine
Vortex samples vigorously for 10-20 s
Spin tubes for 1min to pellet brocken cells
PCR reaction:
10µl 2xMM
2x0.5µl primer
0.4 DMSO
8.6 µl supernatant of cell lysis
Run PCR:
94°C 2min
94°C 30s
55°C 30s
72°C 1min
Repeat step 2 to 4 35x
72°C 5min
Products were loaded on a gel
Bands by 1782bp and 1092bp or 1103bp were expected. Clear products were only visible for the ribozyme constructs. That is why yeast transformation was repeated.
▼2015-08-15 PCR
PCR to check if p413-myc-BFP-Ub-GFP is contaminated.
12.5 µl Phusion Master Mix
1.25 µl xxDH_08p413GPD revxx
1.25 µl xxDH_07p413GPD fwdxx
5µl p413 empty or with protein construct
Add water to 25µl
Program:
97°C 5min
97°C 30sec
52°C 30sec
72°C 1.5min
Step 2 to 4 35 times
72°C 3min
4°C for ever
Results: The sample with p413 with protein construct is not contaminated.
▼2015-08-15 PCR optimization for the p415 series of plasmids
To solve the problems observed with the p415-GPD plamid in PCR reactions we tried to set up an reaction with 1M betaine and 5% DMSO. This mixture has proved to be capable to solve PCR problems with ribozyme secondary structures. Therefore we hoped to solve the issues involving p415-GPD
Setup in 50 µL reactions:
|
|
Volume |
|
2x Phusion Flash PCR Mastermix |
25 µL |
|
Template DNA |
1 µL |
|
fwd. Primer (DH07) |
2.5 µL |
|
rev. Primer (DH08) |
2.5 µL |
|
ddH2O |
6.5 µL |
|
Betaine (5 M) |
10 µL |
|
DMSO |
2.5 µL |
Annealing temperatures were varied in a gradient.
|
# |
Temperature |
|
1 |
72 °C |
|
2 |
68.8 °C |
|
3 |
62.0 °C |
|
4 |
59.4 °C |
|
5 |
56 °C |
Program (Phusion):
|
2 min |
98 °C |
|
|
35x |
15 s |
98 °C |
|
20 s |
Gradient |
|
|
2 min 20 s |
72 °C |
|
|
5 min |
72 °C |
|
|
infinite |
4 °C |
|
Analysis on a 1% agarose gel showed for the reactions with annealing at 62 °C, 59.4 °C and 56 °C the presence of a band at the expected size of ~7kb. The reaction with annealing at 62 °C showed not only the clearest appearance but also the highest yield for the wanted product.
▼2015-08-16 Test digest of received plasmids
To verify we were given valid plasmids from Ilia, we performed a test digest with BsaI and EcoRI. These two enzymes should produce, according to a Geneious virtual digest, a restriction fragment pattern easily distinguished.
To be analyzed:
In DNA minipreps: p413-ADH (#1/#2), p413-cyc1, p413-Gal1, p413-TEF (#1/#2), p415-ADH (#1/#2), p415-cyc1, p415-Gal1, p415-TEF (#1/#2)
Reactions were set up as following:
|
|
Volume |
|
Miniprep DNA from Plasmids |
1 µL |
|
BsaI-HF (NEB) |
0.2 µL |
|
EcoRI-HF (NEB) |
0.2 µL |
|
CutSmart Buffer (NEB) |
1 µL |
|
ddH2O |
7.6 µL |
After digestion for 1 min at 37 °C the reaction mixture was analyzed via 1% agarose gel.
The restriction fragments appeared with sizes according to the expected fragments. Yet for the p413/p415-TEF digestions it appeared that there has been a swap between a p413-TEF and a p415-TEF sample. Before use of a p41x-TEF it is necessary to recheck for the correct backbone.
▼2015-08-16 PCR optimization for BFP site-directed mutagenesis
The SalI site in the BFP of our protein construct (negative and myc-tag positive) has to be removed, to allow for easy recloning into a library of p413 Vectors with different strength promoters via BamHI and SalI.
To find the perfect conditions we resorted to PCR optimization.
Temperature conditions: 72 °C, 66 °C, 60 °C
Polymerases: 2x Phusion Flash PCR Mastermix (Thermo), Q5 2x High-Fidelity Mastermix (NEB)
Buffer solution: Standard Mastermix conditions or addition of 1 M Betaine and 5 % DMSO to counter secondary structure behavior.
Standard buffer conditions:
|
|
Volume |
|
2x Polymerase Mastermix |
5 µL |
|
Template DNA |
0.2 µL |
|
fwd. Primer (DH61) |
0.5 µL |
|
rev. Primer (DH62) |
0.5 µL |
|
ddH2O |
3.8 µL |
DMSO/Betaine buffer conditions:
|
|
Volume |
|
2x Polymerase Mastermix |
5 µL |
|
Template DNA |
0.2 µL |
|
fwd. Primer (DH61) |
0.5 µL |
|
rev. Primer (DH62) |
0.5 µL |
|
ddH2O |
1.3 µL |
|
Betaine (5 M) |
2 µL |
|
DMSO |
0.5 µL |
Reactions were prepared as mastermixes and distributed into 0.2 ml PCR tubes. Cyclinge programs were chosen differently for each polymerase and according to manufacturer’s guideline.
Program (Phusion Flash):
|
15 s |
98 °C |
|
|
35x |
5 s |
98 °C |
|
5 s |
Gradient (72 °C, 66 °C, 60 °C) |
|
|
2 min |
72 °C |
|
|
5 min |
72 °C |
|
|
infinite |
4 °C |
|
Program (Q5):
|
1 min |
98 °C |
|
|
35x |
10 |
98 °C |
|
20 s |
Gradient (72 °C, 66 °C, 60 °C) |
|
|
4 min |
72 °C |
|
|
5 min |
72 °C |
|
|
infinite |
4 °C |
|
The reactions were then mixed with NEB Purple Loading dye and analyzed on a 1% Agarose Gel.
The strongest and most defined bands, to be expected around 7.5 kb were observed with Phusion Flash polymerase in Mastermix buffer at 66° C annealing temperature. We will therefore try to perform the site directed mutagenesis under these conditions.