Team:Freiburg/Labjournals/Cellfree/July

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Labjournal Cellfree July

Fr, 31.07.2015

  • pIG15_105 PCR products were extracted from gel and purified with Qiagen. The resulting DNA showed impurities around 230 nm in repeated measurements in the NanoDrop.

  • → resulting DNA was not used for expression
  • HA-GFP-His6-His6 PCR product was retested in a control gel with a 20 µl sample. The resulting band hints at low DNA concentration
  • pIG15_105 PCRs from 28.07. and 29.07. were also retested by loading 20 µl in a gel. Since again no bands showed up, the PCRs should be repeated using smaller aliquots.

Thu, 30.07.2015

  • PCR purification of pIG15_104 and HA-GFP-His6-His6
  • control gel of HA-GFP-His6-His6

  • after gel extraction DNA concentrations in 22 µl ddH2O with two columns per PCR were a little too low for expression. Therefore the Zymo DNA Concentrator Kit was used to pool both tubes to a total elution volume of 15 µl. After concentrating the DNA concentration diminished to 189.6 ng/µl (HA-GFP-His6-His6) and 107.6 ng/µl (pIG15_104). A second elution did no longer contain clean DNA.
  • Both PCRs have to be repeated.

Careful when using the DNA concentrator kit. It doesn't always work.

Repetition of PCR for cell-free expression template of pIG15_104, pIG15_105 and HA-GFP-His6-His6

  • Both PCRs are performed in 5 x 100 µl reactions with pre-purified PCR product as template (5,2 ng/µl (104) 4,2 ng/µl (GFP)) in 40 cycles
  • All PCRs should be purified on 4 columns and eluted in 10 µl of water.
  • program HA-GFP-His6-His6
Temperature °C duration
985 min
9820 sec
6920 sec
72 30 sec
72 5
4 infinite
  • program pIG15_104
Temperature °C duration
985 min
9820 sec
52 °C 20 sec
72 20 sec
72 5
4 infinite
  • Master Mix
Component amount [µl]
buffer - Phusion HF220
dNTPs55
Primer forward 44 each
Primer reverse 44 each
Phusion Polymerase 1 per tube
Template (pre-purified) <250 ng 1 per tube
DMSO 27.5
H2O to 100µl each 588.5
  • pIG15_105 PCR will be performed in 3 x 25 µl reactions in a gradient from 52 to 54 °C
Temperature °C Sample # Column
52,0 1 1
53,1 2 7
53,9 3 11
Temperature °C duration
985 min
9820 sec
gradient 52 to 54 *20 sec
72 40 sec
72 5
4 infinite

*a mistake in programming occurred. because the cycler was still in touchdown mode, the first 10 cycles ran a gradient followed by 25 cycles at 52°C for all samples.

  • Master Mix
Component amount [µl]
buffer - Phusion HF16.5
dNTPs4.125
Primer forward 6.6
Primer reverse 6.6
Phusion Polymerase 0.5 per tube
Template (pre-purified) <250 ng 1.65
DMSO 2
H2O to 100µl each 44.2
  • control gel showed no band for HA-GFP-His6-His6 (concentration was probably too low).

Wed, 29.07.2015

  • Touchdown PCR for high yield templates of pIG15_105:
    • PCR was performed in 5 x 100 µl reactions with pre-purified PCR product as template (1,93 ng/µl (105)) in 40 cycles
  • Program pIG15_105
Temperature °C duration
985 min
9820 sec
52°C - touchdown from 62 20 sec
72 70 sec
72 5
4 infinite
  • Master Mix
Component amount [µl]
buffer - Phusion HF110
dNTPs27.5
Primer forward 44
Primer reverse 44
Phusion Polymerase 1 per tube
Template (pre-purified) <250 ng 11
DMSO 13.75
H2O to 100µl each 294.25
  • control gel
  • still no product. PCR protocol has to be re-evaluated

Generating PCR template for GFP-Expression
  • Using oQE primer forward and reverse, a HA-GFP-His6-His6 template will be amplified, purified via Gel purification and used as template for a second PCR of pure linear expression template
  • the PCR will be performed in 6 x 25 µl reactions with a gradient from 66 °C to 70 °C
  • the PCR product is expected to have 792 bp
Temperature °C Sample # Column
66,0 1 2
66,6 2 4
67,6 3 6
68,6 4 8
69,5 5 10
70,0 6 12
Temperature °C duration
985 min
9820 sec
gradient 66°C to 70 °C 20 sec
72 30 sec
72 5
4 infinite
  • Master Mix
Component amount [µl]
buffer - Phusion HF33
dNTPs8.25
Primer forward 13.2
Primer reverse 13.2
Phusion Polymerase 0.5 per tube
Template (pre-purified) <250 ng 3.3
DMSO 4.125
H2O to 100µl each 88.275
  • control gel

  • all bands were cut out and pooled for gel extraction
  • PCR for high yield templates of pQE_HA-GFP-His6-His6:
    • PCR was performed in 5 x 100 µl reactions with pre-purified PCR product as template (4,2 ng/µl (1:10 dilution)) in 40 cycles
  • Program
Temperature °C duration
985 min
9820 sec
69 20 sec
72 30 sec
72 5
4 infinite
  • Master Mix
Component amount [µl]
buffer - Phusion HF110
dNTPs27.5
Primer forward 44
Primer reverse 44
Phusion Polymerase 1 per tube
Template (pre-purified) <250 ng 11
DMSO 13.75
H2O to 100µl each 294.25
Dot Blot for Cellfree Expression from Mon, 27.06.15

samples:

  • pIG15_104 (His&Spy), pIG15_105(His&Halo), no DNA (neg control) Promega S30
  • pIG15_104 (His&Spy), pIG15_105(His&Halo) Bernhard lysate + Promega kit (amino acids & premix)
  • pIG15_104 (His&Spy), pIG15_105(His&Halo) Promega TNT 7 Quick Translation and Transcription system
B+P 104 B+P 105 P (S30) 104 P (S30) 105
P (S30) no DNA P (TNT) 104 P (TNT) 105

Tue, 28.07.2015

  • Touchdown PCR for high yield templates of pIG15_104 and pIG15_105:
    • PCR was performed in 5 x 100 µl reactions each with pre-purified PCR product as template (5,2 ng/µl (104) and 1,93 ng/µl (105)) in 10 + 25 cycles
  • Program pIG15_105
Temperature °C duration
985 min
9820 sec
52°C* - touchdown from 62 20 sec
72 70 sec
72 5
4 infinite
  • Program pIG15_104
Temperature °C duration
985 min
9820 sec
52°C - touchdown from 62 20 sec
72 40 sec
72 5
4 infinite
  • Master Mix
Component amount [µl]
buffer - Phusion HF220
dNTPs55
Primer forward 88
Primer reverse 88
Phusion Polymerase 1 per tube
Template (pre-purified) <250 ng 1 per tube
DMSO 27.5
H2O to 100µl each 588.5

  • PCR for 104 looks as expected and will be purified for further use, PCR for 105 has to be repeated.
  • Because the first control gel did not show a ladder, a second gel electrophoresis was performed.

*a mistake in programming the cycler occurred and the annealing temperature was stuck at 62°C after touchdown.

Cellfree expression from yesterday was analyzed under a fluorescence microsope
  • YFP filter set showed no visible fluorescence
  • GFP filter set showed fluorescence in negative control and the combination of Bernhard lysate and Promega kit. Fluorescence in the negative control was less compared to the background.

Mon, 27.07.2015

New cellfree expression

  • Expression of pIG15_104 and pIG15_105 with Promega S30 E.coli Kit (1,3 µg DNA template)
  • Expression of pIG15_104 and pIG15_105 with Promega TNT 7 Quick Translation and Transcription system (1,2 µg DNA template)
  • Expression with Promega S30 E.coli Kit where Bernhard Lysate was used instead of the included S30 lysate (1,3 µg DNA template)
    • Expression of selfmade Luciferase-DNA from koko
    • Expression of pIG15_104 and pIG15_105
  • reactions run for 2 hours in the thermomixer at 30 °C on lowest possible shaking speed
  • afterward reactions wer pipetted into the 96 well plate(White)
  • Luciferase were measured by pipetting 25 µl of Expression mix and 25µl of dilution reagent together in the well
  • Pipetting scheme:
1 2 3 4
E Luciferse with B+P pIG15_104 with B+P pIG15_105 with B+P negative control with Promega
F pIG15_104 with Promega pIG15_105 with Promega
G pIG15_104 withTNT 7 Quick translation and TranscriptionpIG15_105 with TNT 7 Quick translation and Transcription

Thu, 23.07.2015

  • PCR for more pure template of pIG15_105:
    • PCR was performed in 7 x 25 µl reactions with pre-purified PCR product as template (1,93 ng/µl (105)) in 40 cycles
Temperature °C Sample # Column
52,2 1 2
52,9 2 4
53,4 3 5
54,7 4 7
55,3 5 8
56,3 6 10
56,8 7 12
  • Program
Temperature °C duration
985 min
9820 sec
52°C - 57°C 20 sec
72 70 sec
72 5
4 infinite
  • Master mix
Component amount [µl]
buffer - Phusion HF 30,5
dNTPs7,7
Primer forward 12,6
Primer reverse 12,6
Phusion Polymerase 0,5 per tube
Template <250 ng 4,2
DMSO 4
H2O to 183 µl 107,9
  • Touchdown PCR for higher yield of pIG15_104:
    • PCR was performed in 3 x 100 µl reactions with pre-purified PCR product as template (5,2 ng/µl (105)) in 10 + 25 cycles
Temperature °C duration
985 min
9820 sec
52°C - touchdown 20 sec
72 70 sec
72 5
4 infinite
  • Master mix
Component amount [µl]
buffer - Phusion HF76
dNTPs18,5
Primer forward 19
Primer reverse 19
Phusion Polymerase 2 per tube
Template (pre-purified) <250 ng 1 per tube
DMSO 7,6
H2O to 100µl each 202,4
  • control gel, followed by PCR purification

Sun, 19.07.2015

  • Cell free Expression with the Promega Kit and Berhard Mix
    • reactions run for 2 hours in the thermomixer at 30 °C on lowest possible shaking speed
    • afterward reactions wer pipetted into the 96 well plate(White)
    • Luciferase were measured by pipetting 25 µl of Expression mix and 25µl of dilution reagent together in the well
    • Reactions:
      • Luciferase from the Promega Kit in Berhhard mix
      • purified pIG15_104 in Bernhard mix
      • purified pIG15_104 in Promega mix
      • negative control with Berhard control
  • PCR for more pure template of pIG15_105:
    • PCR was performed in 3 x 100 µl reactions each with pre-purified PCR product as template (1,93 ng/µl (105)) in 40 cycles
Temperature °C duration
985 min
9810 sec
52°C 20 sec
72 70 sec
72 5
4 infinite
  • Master mix
Component amount [µl]
buffer - Phusion HF63
dNTPs12,6
Primer forward 15,75
Primer reverse 15,75
Phusion Polymerase 2 per tube
Template <250 ng 6,3
DMSO 6,3
H2O to 50µl 204,3

Fri, 17.07.2015

  • PCR for even more template of pIG15_104 and pIG15_105:
    • PCR was performed in 3 x 100 µl reactions each with pre-purified PCR product as template (1:10 dilution 5,2 ng/µl(104) and 3,1 ng/µl (105)) in 40 cycles
Temperature °C duration
985 min
9810 sec
52°C 20 sec
72 40 sec for pIG15_104, 70 sec for pIG15_105
72 5
4 infinite
  • Master mix
Component amount [µl]
buffer - Phusion HF126
dNTPs25,2
Primer forward 31,5
Primer reverse 31,5
Phusion Polymerase 2 per tube
Template <250 ng 2 per tube
DMSO 12,6
H2O to 50µl 367,8

Thu, 16.07.2015

  • Synthesis of Luciferase Control with the Promega Kit
  1. Synthesize unlabeled luciferase using the following reaction:
Component Volume
pBEST luc™ DNA 8μl
Complete Amino Acid Mixture* (mix before use) 5μl
S30 Premix Without Amino Acids (mix gently before use)20μl
S30 Extract, Linear (mix gently before use) 15μl
Nuclease-Free Water to a final volume of 50μl
  • one negative and one positive control wer pipetted
  • to the negative control 8ml water was added instead of DNA
  • DECEP water was used in the reactions

Water purity is extremely important.

  1. vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the reaction mixture to the bottom of the tube.
  2. Incubation in the thermomixer at 37°C for 2 h
  3. Stop the reaction by placing the tubes in an ice bath for 5 minutes.
  4. Prepare a dilution series as follows:
    • At room temperature, add 50μl of Luciferase Dilution Reagent to each of 4 microcentrifuge tubes.
  • PCR for even more template of pIG15_104:
    • PCR will be performed in 3 x 100 µl reactions with pre-purified pIG15_104 PCR product (1:10 dilution 5,2 ng/µl) in 40 cycles
Temperature °C duration
985 min
9810 sec
52°C 20 sec
72 45 sec
72 5
4 infinite
  • Master mix:
Component amount [µl]
buffer - Phusion HF76
dNTPs18,5
Primer forward 19
Primer reverse 19
Phusion Polymerase 1 per tube
Template (pre-purified) <250 ng 1 per tube
DMSO 7,6
H2O to 100µl each 202,4

→ 98 µl MM per Tube 104, 48 µl for tube 105

  • pI15_105-PCR:
    • repetition
    • 50 µl reactions, 30 cycles
    • 1:100 dilution of template (1,6 ng/µl)
Temperature °C duration
985 min
9810 sec
52°C 20 sec
72 70 sec
72 5
4 infinite

Wed, 15.07.2015

  • PCR of pIG15_104: GelEx

Tue, 14.07.2015

  • cloning of pIG15_105
    • miniprep of 8 clones
    • digest with pBST and HindIII (Fermentas)
component amount [µl]
plasmid 5
Fermentas green buffer 2
Hind III 1
PbsT 1
ddH2O 11
  • control gel:

  • 1.3 and 2.3 were sent for sequencing
  • pI15_104-PCR: repetition
    • control gel showed the right bands

Mon, 13.07.2015

  • cloning of pIG15_105
    • 8 clones were picked
    • 4 clones from each plate

Sun, 12.07.2015

  • cloning of pIG15_105
    • new ligation and Transformation of digested pIG15_104 (HindIII and pBST ) with gelEX from Halo-PCR
    • control with backbone without insert
    • One ligation with help of exel calculator
    • one ligation with so much DNA that no water was needed
  • pI15_104-PCR:
    • repetition
    • two 50 µl reactions, 30 cycles
Temperature °C duration
985 min
9810 sec
52°C 20 sec
72 40 sec
72 5
4 infinite
  • Master mix
buffer - Phusion HF21
dNTPs4,2
Primer forward 5,25
Primer reverse 5,25
Phusion Polymerase 0,5 per tube
Template <250 ng 2,1
DMSO 2,1
H2O to 50µl 64,05

Thu, 09.07.2015

  • PCR for linear templates of pIG15_104 and HA-GFP-His2 showed no bands on gel. Will be repeated sunday. Therefore expression was performed with circular pIG15_104 plasmid
  • Platereader experiment at AG Roth/Diengel together with Norman
    • Use of 4 different mixes: A) One from Norman that he regularly uses („RTS-Kit“), F) Koko's lysate + reaction mix after EMBL, G) Bernhard's lysate + reaction mix after Bernhard and H) the commercial Promega S30 extract Kit for linear templates.
    • reaction size: 50µl each (lysate+reactionmix+dna+h20)
    • in 96well plate, non adhesive material
    • overlayed with each 20 µl of mineral oil to prevent drying out
  • Setup:
x 1 2 3 4 5 6
A neg: water neg: lysate RTS + no DNA RTS + pIG15_104 RTS + (his)2 GFP linear RTS + pEQ60 GFP
.… … … … … …
F Koko + no DNA Koko + pIG15_104 Koko + (his)2 GFP linear Koko + pEQ60 GFP
G Bernhard + no DNA Bernhard + pIG15_104 Bernhard + (his)2 GFP linear Bernhard + pEQ60 GFP
H Promega + no DNA Promega + pIG15_104 Promega + (his)2 GFP linear Promega + pEQ60 GFP
  • reaction mix was prepared first, then the lysate was added and the DNA came last.
  • reaction was performed for 2h at 30°C as Norman recommended it
  • Results:

  • absolutely no expression with any of our kits, not even the Promega one.
  • Norman's RTS kit performed nicely: it shows we should have to expect fluorescence in the ten-thousands after minutes.
  • no idea yet why nothing worked

Wed, 08.07.2015

  • Cloning of pIG15_105:
    • Control gel was empty, probably due to low DNA concentrations
    • Replated pIG15_105 was transferred to 4 liquid colonies (5ml, LB-Cml) to incubate over day and pellet at night.
  • Cellfree expression platereader 2 - linear templates
    • For the next cellfree expression, linear templates are prepared by PCR
      • HA-GFP-His2 will be amplified with the primers pQEfwd and pQErev (provided by AG Roth); product will be 512 bp
      • pIG15_104 will be amplified with oIG15_s001 and oIG15_s003; product will be 1396 bp
      • both PCRs will be performed in triplicates with Phusion Polymerase and purified via gel extraction

* HA-GFP-His2-PCR:

Temperature °C duration
985 min
9810 sec
68°C 20 sec
72 20 sec
72 5
4 infinite
  • pI15_104-PCR:
Temperature °C duration
985 min
9810 sec
51°C 20 sec
72 40 sec
72 5
4 infinite
  • both:
buffer - Phusion HF10
dNTPs2
Primer forward 2,5
Primer reverse 2,5
Phusion Polymerase 0,5
Template <250 ng 1
H2O to 25µl 31,5

→ gel control was empty

Tue, 07.07.2015

  • Cloning of pIG15_105:
    • Control plate of religated pIG15_104 has a lot less colonies than plate with pIG15_105
    • Replating of 4 clones of pIG15_105, incubated over day
    • Miniprep of over-day pIG15_105 lead to poor concentrations between 28 ng/µl and 60 ng/µl
    • Control digest (10 µl)
component amount [µl]
plasmid (200-500 ng) 7
Fermentas green buffer 1
Hind III 1
PbsT 1
ddH2O 0
  • heat inactivated at 80 °C for 10 minutes and kept @-20°C for control gel the next morning.

Mon, 06.07.2015

  • Cloning of pIG15_105:
    • Miniprep of Halotag-Pcr in pJET
    • fast digest of pIG15_104 with Fermentas HindIII und BpsTI

  • fast digest of Halo-PCr in pJEt with Fermentas HindIII und BpsTI

  
  * ligation of empty backbone as control
  * ligation of pIG15_104 and Halo-Tag
  * Transformation: everything plated onto Cml plates, incubated o.N.

Sun, 05.07.2015

  • cloning of pIG15_105:
    • 2 liquid cultures per plate in 5ml LB-Amp to incubate over day and perform a miniprep in the evening

Sat, 04.07.2015

  • cloning of pIG15_105:
    • To clone HALO-tag into corresponding vectors, a gradient PCR was performed on template vector (AG Roth) with oIG105 and oIG106. The resulting fragment should be 931bp. A gradient was done using 8 reaction conditions from 50-60 degrees Celsius. PCR was performed with 32 cycles and 1 min elongation time.
      • Correct bands visible for all reactions, except negative control. Fragments of the three most specific reaction temperatures excised, gel extracted and blunt end ligated into the pJET1.2 blunt end cloning vector (Thermo Fischer, Darmstadt). Selection on Amp+ plates (50 µl and 100 µl of cells spread onto two plates). Negative control was set up without PCR fragment.

Fri, 03.07.2015

  • Repetition of Halo-pcr performed exactly as on 13.06.
  • Halo PCR did not work

  • 1. platereader experiment
    • 25µl reactions
    • Plate Type:96 WELL PLATE, cell culture plates translucent untreted, ploypropylen, flat bottom
    • Set Temperature: Setpoint 37 °C
    • Shake: Medium for 1:00 in the beginning
    • Start –>end Kinetic [Run 4:00:00, Interval 0:05:00]
    • Fluorecence intensity: tYFP 509–>528, GFP: 485–> 510 (Range 9)
    • Pipetting sheme:
    • double marked means that both DNA and water was added
    • red script: Mastermix was added
    • mastermix wasn't sufficient –> add more pipetting error
    • Control for just lysate was forgotten
  • first DNA and water was pipetted into every well
    • If not specified 350 ng DNA was used in the reaction
    • afterward the mastermix was added and layered with 20µl mineral oil
    • everything was done on ice, but pipetting lasted 4 hours (faster method needed)
    • After measurement the whole plate was frozen @ -80 °C
      • many wells showed white crescent
      • no visible fluorecence
  • Minipreps of
    • pQE HA-GFP-2xHis6 (210ng/µl)
    • pQE HA-GFP-His6 (170ng/µl)
    • pQE Ha-GFP (130ng/µl)
    • pQE-60 GP-His (770ng/µl)
  • cloning of pIG15_105:
    • Gel from HALO-Tag PCR from 2.7
      • only barely visible band at the right size → repeat of PCR

  • Repetition of Halo-pcr performed exactly as on 13.06.
  • Halo PCR did not work

  • pJet with HAlO-PCR produkt from 2.7
    • Plate with Halo is empty
    • plate with empty ligation shows clones, but not enough for a normal pJet Cloning –> repetition to check for a contamination of the pJET vector

Thu, 02.07.2015

  • Cloning of pIG15_105:
    • Sequencing of pIG15_105 wasn't positive –> still spy tag inside of vector –> problems with AflII
      • New try with fermentas Enzymes and fast digest needed
  • new test digest of pJET with HALO due to barely visible bands yesterday
component amount [µl]
plasmid (concentration all around 20 ng/µl)5
CutSmart buffer 2
Hind III HF 1
PbsT 1
ddH2O 11

* test digest of eight mini preps (Mastermix)

  • gel of fast digest
    • insert picture here
  • new blunt end ligation of pJET + Halo: according to protocol
quantity/µlingredientcontrol
1ligation fragment (10 ng/µl) -
0,5pJet 0,5
1T4 ligase 1
52xbuffer 5
2,5H2O 2,5
  • incubate for 5 Min @ RT
  • do a Transformation with 5 µl in E. coli TOP10, plated everything
  • keep the rest at 4°C as backup until transformation worked
  • Retrafo of diffent plasmids that were obtained from AG Roth
    • pQE HA-GFP-2xHis6
    • pQE HA-GFP-His6
    • pQE Ha-GFP
      • these plasmids were extensivly tested at AG Roth and show very good expresssion levels
      • These plasmis will be used as additional control (esp. HA-tag seems ta be able to increse yield 10X)
    • Additionally several primers for the creation of linear fragments from the pQE vectors were obtained
      • Amino primer for and rev
      • Primer without aminogroup for cellfree testing
      • Primer with CY3 for testing the binding to surface
      • Primer with TUS/Ter as a binding control
  • miniprep of cultures from yesterday
    • 2 x pQE-60 GFP-His(134 ng/µl but difficulties with dirth in the 220 nm area in one sample (marked)/130ng/µl)
    • 2 x pIG15_104 (160ng/µl / 164ng/µl)
  • repetition of halo-pcr performed exactly as on 13.06. → freezer

Wed, 01.07.2015

  • overnight culture for mini prep (so that enough plasmid is in storage for plate reade experiment)
    • 2 x pQE-60 GFP-His
    • 2 x pIG15_104
  • CfEX GFP from the 2. Cellfree raction was send for measuring in the irif
  • test digest of Halo-tag in pJet and pIG15_105
    • Bands on slightly wrong height –> send for sequencing with s003