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Labjournal Cellfree May

Sat, 30.05.2015:

Gel-Extraction using Qiagen Qiaquick Kit, according to protocol, elution in water

Quantification of DNA in NanoDrop:

component	concentration [ng/µl] in EB-buffer elution	concentration [ng/µl] in ddH2O elution	mean concentration [ng/µl]
pIG15_001 backbone	2,1	1,4	1,75
pIG15_001 insert	3,5	55
pJET backbone	3,7	1,4
pJET insert (=gBlock)	13,3	16,1	14,7

both elution fractions were combined and the concentrations were averaged for further calculations.

Re-ligation of pIG15_001 backbone with gBlock insert:

component	amount [µl]
plasmid backbone (c= 1,7ng/µl)	29,41 (= 50ng)
plasmid insert (c=14,7ng/µl)	13,6 (=200ng)
T4 DNA Ligase buffer	2 (+ 2 after 30min)
T4 DNA Ligase	1

Ligation performed at RT for 1hr.

Transformation of ligated product into competent E.coli

- 24µl plasmid DNA added to 100 µl aliquot of TOP10 cells

- incubation, 10 min, on ice

- 45 sec heat-shock at 42°C

- incubation, 2 min, on ice

- addition of 900µl Lb

- incubation, 1.5h, 37°C, 700rpm

- plating pellet on Cmp-Agar

- incubation, over night, 37°C

Transformation of overnight-ligation from Fri, 29.05 in TOP10 cells

- same protocol as above

Fri, 29.05.2015

Re-ligation of backbone and insert from gel extraction:

component	amount [µl]
plasmid backbone (c=9,3ng/µl)	3 (= 27,9ng)
plasmid insert (c=2,8ng/µl)	16 (=44,8ng)
T4 DNA Ligase buffer	2
T4 DNA Ligase	1

A high insert-to-backbone ratio was used to get higher ligation probability.

The ligation is performed at 4°C over night.

Test digestion of pIG15_001 and pJET + gBlock construct

component	amount [µl]
pIG15_001
plasmid	2
phosphatase buffer	2
CutSmart buffer	2
Xba1	1
Spe1	1
Antarctic phosphatase	1
ddH2O to 20µl	11

component	amount [µl]
pJET
plasmid	2
CutSmart buffer	2
Xba1	1
Spe1	1
ddH2O to 20µl	14

simultaneous dephosphorylation during incubation (1hr, 37°C, 0rpm)

gel electrophoresis of digestion in 1% agarose showed two clear bands in each construct:

The DNA-ladder in both runs is not visible because it has not been stained with Midori Green Direct, like the samples were.

The visible bands were then cut out and stored at - 20°C.

Thu, 28.05.2015

Validation of cloning efficiency due to not getting colonies on chloramphenicol plates.

digestion of pIG15_001 and pJET (containing gBlock) with Not1 HF

component	amount [µl]
pIG15_001
plasmid	10
phosphatase buffer	2
CutSmart buffer	2
Not1 HF	1
Antarctic phosphatase	1
ddH2O to 20µl	4

component	amount [µl]
pJET
plasmid	2
CutSmart buffer	2
Not1 HF	1
ddH2O to 20µl	15

simultaneous dephosphorylation during incubation (1hr, 37°C, 300rpm)
gel electrophoresis of digested products in 1% gel

expected fragment size:

pIg15_001	pJET
2070 bp = backbone	2974bp = backbone
	1080bp = insert

20. - 27.05.2015

successful amplification of IRES sequence from Stanford for eukaryotic expression system
- blunt end ligation in pJET
- plate almost overgrown

lots of unsuccessful cloning of pSB1C3 with G block containing new initiation cassete, no lacI and t7 promotor
- lots of empty plates
- probably due to unsuccesful ligation and therefore no chloramphenicol reistance in cells

Wed, 13.05.2015

package from AG Bernhard from Frankfurt arrived, containing 17x 350µl of CFES with T7RNAP.
stored in -80°C freezer: upper compartment, pink plastic box

Fri, 08.05.2015

gel extraction of PCR product from 07.05. using the QIAquick Kit by Qiagen
changes to QIAquick protocol: Elution with 30µl H2O instead of elution buffer
verification in NanoDrop showed no product (=3ng/µl)
was Koko's first GelEx; unknown error

Thu, 07.05.2015

Construction of prokaryotic cell free expression vector:
no clones on gibson plate
test whether Gibson was successfull/ where the mistake occurred:
- PCR for entire insert (016f 010r)
- fragment size 2020 bp
- total reaction volume: 25 µl

component	amount [µl]
buffer - Phusion HF	5
dNTPs	0,5
Primer forward	1,25
Primer reverse	1,25
Phusion Polymerase	0,25
Template <250 ng	0,5
H2O to 25µl	16,25

Temperature °C	duration
98	5 min
98	10 sec
63	20 sec
72	40 sec
72	5
4	infinite

Wed, 06.05.2015

new Gibson attempt for prokarotic vector

insert	size (bp)	concentration (ng/µl)	volume to use (µl)
backbone	2070	5	2,64
t7 term.	114	12,8	0,45
lacI	1082	32,7	1,69
changed fragment	88	40,2	0,21

Transformation of Gibson mix (leftover reation was frozen)
- 300µl of 500µl were plated on a chloramphenicol plate
- incubation at 37°C over night

Tue, 05.05.2015

Gibson fail: no clones on plate even after retrafo –> decicion to start new Gibson
Gibson:

Fragment	size (Bp)	concentration (ng/µl)	volume (µl)
backbone	2070	5	2,7
t7 term.	114	12,8	0,46
lacI	1082	32,7	1,72
changed fragment	88	40,2	0,2

1% gel of PCR product
Gelex with Qiagen kit/ Elution with 30µl 30°C warm water
Product was frozen at -20°C (21,1ng/µl)

Sun, 03.05.2015

no clones on the plate –> desicion to start at the trafo step with frozen gibson product
Retransformation of gibson from 02.05.2015

Sat, 02.05.2015

Construction of prokaryotic cell free expression vector:
PCR with primer oIG15_016f and oIG15_008r from Pet22b+
- Gel (1%) of PCR with primers oIG15_016f and oIG15_008r from pet22b+
- 95 V 1 h 10 min
fragments have the right size –> gel extraction with Quiagen kit (elution with 30 µl Wasser at 30°C)
concentration changes depending on mood of Nanodrop (around 80 ng/µl)

Cloning of pIG15_003:
Gibson:
- pSB1C3 backbone (chloramphenicol resistant)
- + fragment oIG15_016r and oIG15_008r of Pet22b+
- + fragment oIG15_001f and oIG15_002 of Pet22b+ (lac1: 32,7 ng/µl)
- + fragment oIG15_009f and oIG15_010r of Pet22b+ (T7 ter: 32,7ng/µl)
Transformation

Fri, 01.05.2015

Construction of prokaryotic cell free expression vector:
PCR with Primer oIG15_016r and oIG15_008r from Pet22b+
- 2% gel of PCR with primer oIG15_016r and oIG15_008r from Pet22b+
- gel melted –> decision to use 1% gel

Team:Freiburg/Labjournals/Cellfree/May