Team:Potsdam/laborbuch1

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Demonstrate - Gold
Thursday, 10/19/17

sgRNAs:
MS2 sgRNA PP7 sgRNA
Primer 1 IG_C_Q63 IG_C_Q64
Primer 2 IG_C_Q65 IG_C_Q66
Annealing temperature 70 °C 72 °C
size 292 bp 263 bp
Master mix for 6 samples
component volume
Q5 Reaction buffer 30 µL
10 mM 3 µL
Q5 Polymerase 1.5 µL
GC Enhancer 30 µL
ddH2O 64 µL
-21.5 µL Mastermix + 1.25 µL Primer 1 + 1.25 µL Primer 2 + 1 µL template
-(templates: 0.1 ng/µL and 1 ng/µL of the sgRNAs)
-testgel: 2 µL PCR sample+ 8 µL H2O + 2 µ Loading Dye
--> samples were nearly run out of the gel--> but it looks like there is only one band
--> PCR Clean up
--> determination of the concentration:
sgRNA-Ms2: 169.1 ng/µL
sgRNA-PP7: 155.2 ng/µL
--> testgel with 25 ng sgRNA:
-sgRNA bands were at the expected height and no additional bands were visible
-the bands show also an expected intensity
-Opening of the P4 plasmid
-->the GXL Polymerase was used
component volume
5x GXL buffer 10 µL
10 mM dNTPs 4 µL
IG_C_Q59 1 µL
IG_C_Q60 1 µL
GXL DNA Polymerase 1 µL
H2O 31 µL
--> 24 µL Mastermix + 1 µL template (P4 clone 44/55, 1 ng/µL)
PCR Program:
temp (°C)
 time
98 °C 10 s
68 °C 7 min 18 s
--> was running over night

Friday, 10/20/17

-Opening of the P4 plasmid
-2 µL PCR sample + 2 µL Loading Dye + 8 µL H2O
-> Load on a 0.7 % agarose --> run for 30 min at 120 V
--> one clear band is visible
--> we will do a PCR clean up, but before we digest the samples with DpnI:
--> add 1 c DpnI to the sample--> incubate at 37°C for 80 min--> heat inactivation at 80 °C for 20 min
--> PCR Clean-up and determination of the concentration:
P4 clone 44: 70 ng/µL
P4 clone 55: 83.2 ng/µL
-Preparation of parts for iGEM HQ:
-IAA Fusion from P4:
componente volume
Primer 1 IG_C_Q68
Primer 2 IG_C_Q67
Annealing temperature 63 °C
size 4000 bp
-LacI-dCas9 from P5:
componente volume
Primer 1 IG_C_Q37
Primer 2 IG_C_Q38
Annealing temperature 65 °C
size 5728 bp
-PCR Mastermix for 3 samples:
component volume
5x Q5 reaction buffer 15 µL
10 mM dNTPs 1.5 µL
Primer 1 3.75 µL
Primer 2 3.75 µL
GC Enhancer 15 µL
Q5 DNA Polymerase 32.25 µL
-24 µL Mastermix + 1 µL template
template 1: P4 clone 44
template 2: P4 clone 55
template 3: P5 (1 ng/µL solution from 5.9.17)
--> start PCR programm:
component temp (°C)
 time
Initial Denaturation 98 °C
Denaturation 98 °C 35x
Annealing 63 °C/ 65 °C
Elongation 72 °C
final Elongation 72 °C
--> 2 µL PCR sample + 2 µL Loading Dye + 8 µL water
--> Load on 0.7 % Agarose gel
--> run for 30min at 120 V
--> Purification: PCR clean up for LacI-dCas9, Gel clean up for IAA-Fusion
--> DNA concentration:
-LacIdCas9: 59.1 ng/µL
-IAA-Fusion (P4 clone 44): 39.8 ng/µL
-IAA-Fusion (P4 clone 55): 51.1 ng/µL
--> Digestion:
component volume
template (LacIdCas9/IAA-Fusion (P4 clone 44)/IAA-Fusion (P4 clone 55)) 16 µl
EcoRI-HF 1 µl
PstI 1 µl
NEB buffer 2.1 2 µl
--> incubate at 37°C for 1h
--> Heat inactivation for 20 min at 80 °C
--> Ligation:
-Composition of ligation sample:
component volume
template (digested LacIdCas9/IAA-Fusion (P4 clone 44)/IAA-Fusion (P4 clone 55)) x µl
T4 Ligase
 1 µl
T4 Ligase buffer
 2 µl
pSB1C3 4,75 µl
water to 20 µl
-Needed volumes of template "x":
LacIdCas9: 7.2 µL
IAA-Fusion (P4 clone 44): 7.5 µL
IAA-Fusion (P4 clone 55): 5.9 µL
--> Incubation at 16 °C over night

Monday, 10/21/17

-Transformation of ligation (20.10) LacI dCas9, IAA-Fusion (clone 55) and IAA-Fusion (clone 44) in psB1C3
-5 µl template each in DH5α (25 µl)
-> plated on CAP
-> incubation over night at 37 °C
label: IAA-Fusion clone 55
1: with7,4 µl template
2: with 5,9 µl template

Sunday10/22/17

-Colony PCR of the Transformations from saturday
-LacI-dCAS9 in pSB1C3:
Primer 1 IG_C_Q17
Primer 2 IG_C_Q20
Annealingtemp 53°c
Elongation time 1min 39s
size 1642 bp
-IAA Fusion in pSB1C3
Primer 1 IG_C_Q18
Primer 2 IG_C_Q24
Annealingtemp 52°c
Elongation time 44s
size 725 bp
-Mastermix for 12 samples:
component volume
Quick Load Taq Mastermix 120 µL
Primer 1 4.8 µL
Primer 2 4.8 µL
H2O 110.4 µL
-aliquote 20 µL Mastermix in the tubes and a resuspended clone
10x IAA Fusion (out of P4 clone 44) in pSB1C3 + control
10x IAA Fusion (out of P4 clone 55) in pSB1C3 + control
10x LacIdCAs9 in pSB1C3 + control
--> Start PCR Program
steps temp (°C)
 time
Initial Denaturation 95 °C 30s
Denaturation 95°C 20s
Annealing (52°C/53°C) 30s
Elongation 68°C (44s/1min 39s)
Final Elongation 68°C 5min
--> Load on a 0.7 % Agarose gel
--> Colony PCR for the IAA Fusion was positive --> Preparation of 5 mL liquid culture (CAM) using the clones indicated with red dot
-->Colony PCR of LacIdCas9 was negative, Althought we prepared 5 mL liquid culture (CAM) using 2 clones (indicated with red dot)
(check if negative result is due to PCR or transformation in yeast--> an monday a testdigestion should be conducted)

Monday, 10/23/17

-Minipreps of IAA fusion in pSB1C3 and LacI-dCas9 in pSB1C3 (22.10.) according to protocol
concentrations (IAA fusion P4 (44))
 concentrations (IAA fusion P4 (55))
c(1) = 662,5 ng/µl c(4) = 466,4 ng/µl
c(2) = 630,9ng/µl c(5) = 518,5 ng/µl
c(3) = 397,5 ng/µl c(7) = 535,3 ng/µl
c(4) = 536,3 ng/µl c(8) = 501,6 ng/µl
c(6) = 604,9 ng/µl c(10) = 410,6 ng/µl
c(7) = 593,4 ng/µl
c(8) = 617,4 ng/µl
c(9) = 520,8ng/µl
c(10) = 537,0 ng/µl
-> Now: decision which of those will besent to iGEM HQ -> DNA has to be dried
-Gycerol stocks of clone 1 and 7 (1:1)
-Test digest of those
component volume
template (miniprep) 2 µl
NEB 2.1 1 µl
EcoRI-HF 0,2 µl
PstI 0,2 µl
H2O 6,6 µl
-incubation for 1h 15 min at 37°
-Test gel (0,7% agarose)
-expected lengths:
IAA fusion: 4300 bp
pSB1C3: 2070 bp
LacI-dCAs9: 5760 bp
-gel run unusual: curved bands, ladder unclear
-Bands not exactly were they should be
-> IAA fusion P4 (44) clones all positiv
-> IAA fusion P4(55) clones 1+2 -> 3 bands (but others positive)
-LacI-dCas9 definetely negative
-PCR for P6
component volume
5x Q5 reaction buffer 5 µl
10 mM dNTPs 0,5 µl
10 µM IG_C_Q69 1,25 µl
10 µM IG_C_Q62 1,25 µl
Q5 Polymerase 0,25 µl
GC Enhancer 5 µl
ddH2O 10,75 µl
Program:
steps temp (°C)
 time cycles
Initial Denaturation 98 °C 80 s
Denaturation 98 °C 10 s 40x
Annealing 72° 30 s
Elongation 72 °C 2:51 min
final Elongation 72 °C 5 min
-Test gel: negative (gel again weird!)
-Repeated gel: negative again (but also ladder looked strange: made new Agarose and discarded the old)
-Repeated PCR (over night)
-Please put on gel tomorrow

Tuesday, 10/24/17

-TestGel of PCR (120V, 0,7%, 30min)
-->all clones negativ => gradient PCR
-gradient PCR of IG_C_P5_I (1ng/µl)
Master Mix:
component volume
5x Q5 reaction buffer 35 µlm
10 mM dNTPs 3,5 µl
10 µM IG_C_Q69 8,75 µl
10 µM IG_C_Q61 8,75 µl
Q5 Polymerase 1,75 µl
GC Enhancer 35 µl
ddH2O 75,25 µl
Program:
steps temp (°C)
 time cycles
Initial Denaturation 98 °C 30 s
Denaturation 98 °C 10 s 40x
Annealing tube 1 = 71,6°C 30s
tube 2 = 69,4°C 30s
tube 3 = 73,8°C 30s
tube 4 = 68,5°C 30s
tube 5 = 74,8°C 30s
Elongation
 72 °C
 2 min 15s
Final elongation
 72 °C
 5 min
-Media for GC-MS-Cultures:
a)
component volume
M9 Minimal salts
 500 ml
20% Glucose
 20 ml
MgSO4 2 ml
CaCl2 1 ml
Trace element mix
 1 ml
10g/L Tryptophan
 400 ml
water 76 ml
b)
component volume
M9 Minimal salts
 500 ml
20% Glucose
 20 ml
MgSO4 2 ml
CaCl2 1 ml
Trace element mix
 1 ml
water 476 ml
*trace element mix:
component mass
H3BO3 28,6mg
MnCl2*4H2O 18,1mg
ZnSO4*7H2O 2,22mg
Na2MoO4*2H2O 3,9mg
CuSO4*5H2O 0,8mg
Co(NO3)2*6H2O 0,5mg
water to 10 ml
Wednesday, 10/25/17

-gel clean up of LacI-dCas9 (gradient PCR from yesterday)
final concentration: 10,1 ng/µl
-new test digest of the IAA fusions (because gel from october 23rd looked so strange)
-batch and expected sizes see low copy lab book p.132
-Result: all four clones (44 (1), 44 (8), 55 (3) & 55 (7) - they were picked randomly) are positive!
-Help for Berlin: digest of Cap TaC3 3 Fra to gain pSB1C3
component volume
template (Cap TaC3 3 fra) (143,6 ng/µl)
 14 µl
NotI-HF 2 µl
CutSmart 2 µl
water 2 µl
-Transfered some µl of this to in new tube, just in case we still need this

Thursday, 10/26/17

-gibson assembly of LacI-dCas9Q62/Q69 with P4 Q59,Q60
-did it for both P4 clones: 44 and 55
component volume
HiFi DNA Assembly Mix 10 ul
P4 (70 / 83,2 ng/ul) 44: 0,36 ul/ 55: 0,3 uL
LacI-dCas9 (10,1 ng/ul) 5,86 ul
ddH2O 3,78 uL/3,84 uL
-probably won't work because LacI-dCas9 was left on the bench ver night
-incubated at 50°C for 20 min
-transformed into JM109 competent cells
-platend on Amp
-prepared parts for shipping to iEM HQ:
IAA-RBP (K2483000)
TaC1 (K2483005)
TaC3 (K2483006)
Ddx4-YFP (K2483007)
-preparing overnight cultures of E-coli for GC-MS measurements:
-all cultures in minimal media with or without tryptophan
8 x JM109 cells without plasmid (4x with and 4x without tryptophan)
8 x empty pSB4A5 backbone (4x with and 4x without tryptophan)
8 x media without anythin else (4x with and 4x without tryptophan)
4 x P4 clone 44 (2x with and 2x without tryptophan)
4 x P4 clone 55 (2x with and 2x without tryptophan)

Friday, 10/27/17

-shipping of Parts (prepared on 26.10.17)
-centrifuged and extracted cultures for GC-MS-calculation (inoculated on 26.10.17)
-amplification of LacI-dCas9 with homologous overlabs to P4 with IG_C_Q26 and Q69
-PCR Program of 25.10.17 with 4 diffrent Temperatures ( 66°C-69°C)
-->gel clean up: 137,7 ng/ul

Saturday, 10/28/17

-Gibson assembly of LacI-dCas9 and P4 (clone 44 and 55)
P4 (44)
 P4 (55)
component volume component volume
HiFi DNA Assembly Mix 10 ul HiFi DNA Assembly Mix 10 ul
P4, 70 ng/µl (44) 0,7 µl P4, 83,7 ng/µl (55) 0,6 µl
LacI-dCas9 0,6 µl LacI-dCas9 0,6 µl
ddH2O 8,7 µl ddH2O 8,8 µl
-incubation for 20 min at 50°
-Transformation of both Gibson assemblies into competent JM109
-plated 110 µl of both clones on one Amp plate (plate divided) -> only one Amp plate left!
-Plated the rest on Amp+Trp plates
-> in 37° incubator over night

Sunday, 10/29/17

-Transformation from yesterday (28.10): no colonies