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Cloning

2015/05/01

DpnI digest of the PCR fragments (RE)

2 µl DpnI added to each sample
incubation at 37°C for 60 min
heat inactivation at 80°C for 20 min

PCR clean-up (RE)

used Roche kit
according to the instructions
eluted in 100 µl elution buffer

NanoDrop analysis (RE)

Fragment	concentration [ng/µl]
lacI	32.7
CMV	56.9
WPRE	24.8
T7 promoter	20.6
T7 terminator	12.8
TurboYFP (1)	34.3
TurboYFP (2)	39.3

(high 220 nm absorption in all samples)

Restriction Digests (RE)

TurboYFP:

20 µl TurboYFP PCR product (1)→ 700 ng
0.7 µl of each restriction enzyme (SalI + NotI)
3 µl NEB Buffer 3.1
6.6 µl ddH2O

–> total volume: 30 µl

pET51b+:

2 µl pET51b+ → 100 ng
0.1 µl of each restriction enzyme (SalI + NotI)
2 µl NEB CutSmart buffer
15.8 µl ddH2O

–> total volume: 20 µl

pSB1C3:

4 µl linearized backbone → 100 ng
0.1 µl of each restriction enzyme (EcoRI and SpeI)
2 µl NEB CutSmart Buffer
13.8 µl ddH2O

–> total volume: 20 µl

Incubation at 37°C for 15 min (HF restriction enzymes); Heat inactivation at 80°C for 20 min

Preparation of Gibson Master Mix (RE)

100 µl ISO buffer (5x)
0.2 µl T5 Exonuclease (10 U/µl; NEB)
6.25 µl Phusion Polymerase (2 U/µl; NEB)
50 µl Taq Ligase (40 U/µl; NEB)
218.55 µl ddH2O

–> total volume: 375 µl; 15 µl aliquots stored at -20°C

Ligation of TurboYFP and pET51b+ (RE)

according to wiki protocol

–> pIG15_101

Gibson Assembly (RE)

pIG15_001
pIG15_002 (1x with self-made Gibson-MM, 1x with Gibson-MM from Philipp)
according to wiki protocol

Transformation in E. coli TOP10 (RE)

pIG15_001 (on LB-Cml)
pIG15_002 (2x) (on LB-Cml)
pIG15_101 (on LB-Amp)
according to wiki protocol
15 µl were spread on the plates
o/n at 37°C

2015/05/02

Transformation results (RE)

pIG15_101 –> 3 colonies
pIG15_002 –> 3 colonies
pIG15_001 –> nothing

Repetition of the Trafo (RE)

changes:
2 µl plasmid
30 µl spread on the plates

2015/05/03

Mini-Prep: pIG15_101 and pIG15_002 (RE)

pIG15_101 (3x) and pIG15_002 (2x) are prepped with the PeqLab-Kit
according to the manufacturers protocol
eluted in 50 µl Elution Buffer

Nano-Drop (RE)

construct	concentration [ng/µl]
pIG15_101 (1)	83.3
pIG15_101 (2)	78.1
pIG15_101 (3)	70.2
pIG15_002 (1)	205.5	–> sequencing (2015/05/08): correct
pIG15_002 (2)	201.9	–> sequencing (2015/05/08): correct

Test Digests (RE)

pIG15_101 was digested with SalI-HF and BamHI-HF

0.25 µl of each enzyme
1 µl CutSmart buffer
250 ng plasmid

pIG15_002 was digested with EcoRI-HF and PstI

1 µl of each enzyme
2 µl CutSmart buffer
1 µg plasmid

incubation for 20 min at 37°C; heat inactivation for 20 min at 80°C

Gel Electrophoresis (RE)

1% agarose gel; 120 V for ~45 min

pIG15_101 was not digested as expected, whereas pIG15_002 was digested into the two fragments (~2 kb and ~1.3 kb) which were expected.

2015/05/08

PCR to verify insertion of TYFP in plasmid construct (LS)

Test with 3 plasmids: YFP 1, YFP 2, YFP 3 –> test digest didn't give clear results

PCR with: oIG15_101 + oIG15_102
4x Master Mix

	volume
Buffer	16µl
oIG15_102	4µl
oIG15_102	4µl
dNTPS	1,6µl
DMSO	2,4µl
Phusion HF	0,8µl
Template	0,5µl
H2O	50,7

Cycling

Temperature	time
92°C	180s
92°C	20s
60°C	20s
72°C	20s
72°C	10 min
4°C	infinite

–>22 cycles

Analysis of PCr on 1% agarose gel (RE)

120, ~40 min
no PCR product visible

2015/05/10

Repetition of PCR to verify insertion of TYFP in plasmid construct (LS)

Test with 3 plasmids: YFP 1, YFP 2, YFP 3 –> test digest didn't give clear results
PCR with: oIG15_101 + oIG15_102
10x Master Mix (9x with 10ng template + 9x with 1ng template)

	volume
Buffer	40µl
oIG15_102	10µl
oIG15_102	10µl
dNTPS	4µl
DMSO	6µl
Phusion HF	2µl
Template	0,3/0,03µl
H2O	126,75/127,875

Cycling

Temperature	time
92°C	180s
92°C	20s
60°C*	20s
72°C	20s
72°C	10 min
4°C	infinite

–>gradient from 60°C to 68°C; 30 cycles

Digest (LS)

pET51b+

1 µl SalI-HF
1 µl NotI-HF
2 µl CutSmart buffer
2 µl plasmid (100 ng)
14 µl H2O

TurboYFP (2)

1 µl SalI-HF
1 µl NotI-HF
2 µl CutSmart buffer
14.8 µl plasmid (560 ng)
1.2 µl H2O

–> 15 min at 37°C; heat inactivation 10 min at 80°C

Vector dephosphorylation (LS)

total digest product
1 µl antarctic phosphatase (NEB)
2 µl antarctic phosphatase buffer (NEB)
up to 60µl dH2O

–> 1 h at 37°C

T4 Ligase buffer aliquoted (LS)

–> 16 µl per aliquot; stored at -20°C with the other buffers

2015/05/12

PCR for parts of the pIG expression plasmid

4x Master Mix:

Ingredient	volume [µl]
Phusion GC Buffer	16
Phusion pol	0.8
dNTP Mix	1.6
DMSO	2.4
ddH2o	50.7

+ 1 µl of forward and reverse primer, respectively

fwd	rev	fragment
oIG15_001f	oIG15_002r	lacI
oIG15_007f	oIG15_008r	T7 promoter
oIG15_009f	oIG15_010r	T7 terminator

–> final volume per sample: 20 µl

Cycling conditions as on 2015/05/08

–> analyzed on 1% agarose gel:

image could not be saved on the stick
lacI was amplified
T7 promoter and terminator: no bands on the gel
Marker lane: also nothing seen on the gel…

2015/05/12

Repetition of PCR for LacI, T7 Promotor and T7 terminator (LS)

Same mix and cycling conditions as on 2015/05/08

2015/05/13

Gel-Ex of yesterday's PCR product (LS)

Qiagen kit
eluted in 50 µl
Nanodrop:

Fragment	Concentration [ng/µl]
lacI	14.8
T7 terminator	18.4
T7 promoter	21.8

Gibson (LS)

Gibson trafo into E. coli TOP10 according to the wiki protocol
DNA-Mix: 1.7 µl pSB1C3 (digested), 2.95 µl lacI, 0.19 µl T7 terminator, 0.16 µl T7 promoter
total cells spread on two plates LB(cml)

Transformation of pET51b(+) and pET22b(+) into E.coli T10 (LS)

according to protocol
100 µl each spread on LB(amp)
inoculation of 5 ml LB + Ampicillin each
incubation at 37°C o/n.

2015/05/14

Miniprep (LS)

pET22b+ –> 133,7 ng/µl
pET51 –> 136,1 ng/µl

Inoculation of liquid culture: pIG15_001 (LS)

streaked out 8 colonies from transformation plates on new LB (+chloramphenicol 25µg/ml) plates
inoculation of lb (+chloramphenicol) with same colonies
incubation at 37°C

2015/05/15

PCR for tYFP insert and gBLock insert (LS)

tYFP
insert for pIG15_001 from gBlock

	MM tYFP	MM insert
Buffer	16µl	8µl
oIG15_102	4µl	2µl
oIG15_102	4µl	2µl
dNTPS	1,6µl	0,8µl
DMSO	2,4µl	1,2µl
Phusion HF	0,8µl	0,4µl
Template	0,2µl	0,58µl
H2O	50,7	8µl

PCR program:

step	T[°C]	t[s]
initial denaturation	92	180
denaturation	92	20
annealing	60	20
extension	72	30
final extension	72	600

–>PCR didn't work for tYFP, for the gBlock insert the fragment showed a weak band at the right size, but the concentration was only: 17,1 ng/µl.

Miniprep: 8 clones from pIG15_001 (LS)

clone number	concentration (ng/µl)	test digest(EcoRI + PstI)	comment
1	196,4	insert :)
2	187,4	no insert :(
3	182,1	insert :)
4	215,5	insert :)
5	212,1	insert :)
6	259,2	insert :)	–> sequencing (2015/05/18): correct
7	120,1	(maybe insert…)
8	169,4	insert :)	–> sequencing (2015/05/18): correct

Test digest
5µl	plasmid
0,5µl	EcoRI HF
0,5µl	PstI HF
1µl	CUt Smart
3µl	dH2O

incubation at 37°C, 1h

Something was wrong with the agarose, but the bands are still visible. Clone 2 and 7 did not show a positive test digest, but the rest was fine. Clone 6 and 8 were sent for sequencing, both with correct results.

Gibson Assembly: pIG15_001 (LS)

Gibson Assembly
pSB1C3 (digested with EcoRI and SpeI)	2,36µl
gBlock insert (=T7 promotor without Xba1 restriction site)	0,52µl
T7 terminator	0,65µl
lacI	1,57µl

incubation at 50°C for ~1h
3 min cool down
3 min incubation on ice
transformation (7,5µl) in E.coli Top 10 (according to protocol)
plated cells on LB plate with chloramphenicol

2015/05/16

Gibson Assembly: checking plates (LS)

didn't work, no clones visible on LB-chloramphenicol plates

PCR for tYFP + gBLock T7P

same procedure as last time
really bad gel:
gel-ex:
nearly no result, concentration below 5ng/µl
nothing for the gBLockT7P

2015/05/17

Gibson Assembly: checking plates (LS)

still no clones visible
chucked the plates

repetition of PCR: gBlockT7P and tYFP (LS)

different denaturation temperature (98°C)
again bad gel pic:

[

Repetition of PCR again for tYFP (LS)

slight bands on the gel
this time clean-up from PCR product directly without gel-ex (Roche kit)
again: nothing!!!

Repetition of PCR: gBLock with gradient for annealing temperature: 55°C-65°C (LS)

55°C: nice band
~58°C: slight band
>60°C: nothing

Repetition of PCR: tYFP

PCR worked :)

blunt-end ligation of pJEt and tYFP
Gibson of pSB1C3 and gBLock T7P + T7T+lacI
transformation in E.coli Top 10
plated on LB-Amp/LB-Chloramphenicol
incubation at 37°C

2015/05/19

Gibson Assembly: checking plates (LS)

no colonies on LB-chloramphenicol plates (gBlock T7P in pSB1C3)
colonies on LB-AMp plates for blunt-end ligation of tYFP in pJET
picked 6 clones
incubation (shaking) at 37°C in liquid LB-Amp for ~8h
mini-prep of 4 clones (the other 2 did't grow)

Restriction digest (LS)

pET51 and tYFP (in pJET) with SalI and NotI
additional: dephosphorylation of pET51

ingredient	volume (pJET)	volume (pET51)
DNA	5µl	5µl
SalIHF	1µl	1µl
NotI	1µl	1µl
NEB buffer 3.1	5µl	5µl
dH2O	38µl	32µl
Antarctic phosphatase	–	1µl
Antarctic phosphatase buffer	–	5µl

incubation at 37°C for 1h
gel-run of digest
digest didn't work

2015/05/20

Gibson Assembly: gBLock T7P in pSB1C3 + T7T +LacI (LS)

still no clones on LB-chloramphenicol plates
Gibson didn't work

PCR: Antigen 4 (DV) and Antigen 9 (HSV1) (LS)

primer: * oIG15_401 and oIG15_402
- oIG15_801 and oIG15_802

ingredient	pIG15_402 (173ng/µl)	pIG15_802 (214ng/µl)
Buffer	12	12
Primer	3	3
Phusion	0,6	0,6
dNTPs	1,2	1,2
DMSO	1,8	1,8
dH2O	38,23	38,26
Template	0,17	0,14

cycle:

temperature	time
92°C	3min
92°C	20s
60°C	20s
72°C	20s
72°C	10 min
4C	infinite

gel-run: Antigen 4(DV) didn't work, Antigen 8 (HSV1)–>gel-ex: concentration: 9,9ng/µl

PCR: 3×50µl gBlock T7P (LS)

ingredient	volume
Buffer	30
Primer	7,5
Phusion	1,5
dNTPs	3
DMSO	4,5
dH2O	95,12
Template	0,88

temperature	time
92°C	3min
92°C	20s
60°C	20s
72°C	20s
72°C	10 min
4C	infinite

analysis on 1% agarose gel/ gel-ex
concentration: 105ng/µl

PCR: Antigen 4(DV) 2×20µl (LS)

ingredient	volume
Buffer	8
Primer	2
Phusion	0,4
dNTPs	0,8
DMSO	1,2
dH2O	25,54
Template	0,06

temperature	time
92°C	3min
92°C	20s
60°C	20s
72°C	20s
72°C	10 min
4C	infinite

analysis on 1% agarose gel/ gel-ex
concentration: 23,4ng/µl

Sequencing (RE)

Spy-tag (2x): oIG15_s001
Spy-catcher (2x): oIG15_s001
pIG15_001 (clone 6 and 8): oIG15_s003
pET51b+: T7minus1 (GATC Standard)

2015/05/21

Digest and dephosphorylation (RE)

pIG15_001 (6) - prokaryotic backbone (BamHI and AflII)
pIG15_002 (1) - eukaryotic backbone (BamHI and AflII)
pSB1A3 - linearized standard backbone (EcoRI and SpeI)

1 µg DNA
2 µl CutSmart Buffer
1 µl of each restriction enzyme
1 µl Antarctic phosphatase
up to 20 µl ddH2O

–> incubated 1 h at 37°C

Clean-up of digested and dephosphorylated plasmids (LS)

Zymo DNA clean-up kit

concentrations
pSB1A3	31.6 ng/µl
pIG15_001	96.1 ng/µl
pIG15_002	92.7 ng/µl

Gibson assembly:pIG15_001, pIG15_403 and pIG15_803 (RE)

pIG15_001 (with pSB1A3 backbone!!!):

Fragment	amount [µl]
pSB1A3	0.43
lacI	3.88
T7 terminator	0.6
T7 promoter (gBlock)	0.1

* pIG15_403:

Fragment	amount [µl]
pIG15_002	0.53
Dengue virus antigen (4)	4.47

* pIG15_803:

Fragment	amount [µl]
pIG15_002	0.56
Herpes simplex antigen (8)	4,44

PCR: Antigen 4 (DV) (LS)

fragment from first PCR had the wrong size
expected band length: ~1000bp
band lenght on gel: between 200 and 300bp

Repetition of PCR: Antigen 4 from different templates (LS)

this time gradient PCR: 55°C to 65°C (annealing temperature)
denaturation temperature: 98°C instead of 92°C to prevent secondary structures of primers

PCR: Antigen 4
	template: pI15_401	template:gBlock DV1
buffer	52µl
oIG15_401f	13µl
oIG15_402r	13µl
dNTPs	5,2µl
DMSO	7,8µl
Phusion Polymerase	2,6µl
template	1,54µl	1,5µl
dH2O	164,86µl	164,9µl

Cycling (25x)
98°C	3min
98°C	20s
55°C-72°C	20s
72°C	20s
4°C	–

PCR products were kept in fridge o/n

2015/05/22

Gel pic from gradient PCR for Antigen 4 (DV1)

Marked red: Bands that were cut out and used for gel-ex clea up.

Gibson: pIG14_403 again (DV1 in pIG15_002) (LS)

pIG15_002 (digested BamHI + AflII): 0,44µl
Antigen 4 (DV1): 3,47µl
added to Gibson Mix
10µl transformed in E.coli Top 10

Transformation: Plasmids with Antigens without Tag in pJET in E.coli Top 10

pIG15_301
PIG15_401
pIG15_501
pIG15_601
pIG15_701
pIG15_801
pIG15_901
pIG15_1001
pIG15_1101

–>transformation according to protocol

Gibson for pIG15_001A and pIG15_803: picked 8 clones each (LS)

on the plate for pIG15_403 were lots of clones, but since we found out that DV1 is ~1000bp and we accidently cloned pIG15_403 with a fragment that was around 300bp long, we didn't use this plate
incubation in LB-media with Amp or Chloramphenicol for around 8h

Miniprep of all clones (pIG15_001A + pIG15_803) (LS)

concentrations:

pIG15_803	[ng/µl]	pIg15_001A	[ng/µl]
1	277,3	1	171,8
2	228,9	2	242,4
3	308,8	3	267,8
4	268,3	4	231,8
5	302,0	5	234,0
6	295,8	6	235,1
7	300,7	7	172,9
8	209,3	8	142,6

2015/05/23

Miniprep of yesterdays trafo (pIG15_301 - pIG15_1101) (RE)

Zymo kit
eluted in 60µl
Nano-Drop:

plasmid	[ng/µl]
pIG15_301	17,4
pIG15_401	40
pIG15_501	34,2
pIG15_601	53,5
pIG15_701	45,3
pIG15_801	12,1
pIG15_901	21,2
pIG15_1001	29,8
pIG15_1101	52,5

Test digest: pIG15_803 + pIG15_001A with BamHI and SpeI (LS)

8 clones for pIg15_803
8 clones for pIG15_001A
digest in 20µl with BamHI HF and SpeI HF

Correct clones: pIG15_803 (6) and (7)/pIG15_001A (4) and (5)

Sequencing

correct clones:
- pIG15_803-6 and pIG15_803-7
- pIG15_001A-4 and pIG15_001A-5

Repetition of transformation from 2015/05/22 (RE)

2015/05/24

Mini-Prep: pJET with Antigen gBlocks (RE)

plasmid	c [ng/µl]
pIG15_301	633
pIG15_401	638
pIG15_501	422
pIG15_601	358
pIG15_701	651
pIG15_801	979
pIG15_901	368
pIG15_1001	305
pIG15_1101	212

Biozym-Kit
eluted in 30 µl dH2O (pIG15_501 in 60 µl)

Mini-Prep: pIg15_403 (DV1 in pSB1C3 backbone with 10x His-tag) (LS)

plasmid	c [ng/µl]
pIG15_403-1	140,3
pIG15_403-2	115,6
pIG15_403-3	119,2
pIG15_403-4	119,9
pIG15_403-5	138,5
pIG15_403-6	168,2
pIG15_403-7	96,5
pIG15_403-8	66,0

test digest with BamHI and PstI

volume	ingredient
5µl	plasmid
1µl	BamHI
1µl	PstI
1µl	Cut Smart Buffer
3µl	dH2O

digest didn't work
repetition: this time in 20µl
didn't work again

Repetition of Gibson for pIG15_403 (RE)

0,53µl digested pIG15_002
4,47l DV1
added to Gibson Mix
transformation in E.coli Top 10 (10µl)

Digest of backbones (LS)

pSB1A3, pIG15_002, pIG15_803-6 and pIG15_803-8
digested with EcoRI and PstI
50µl
incubation at 37°C for 1 h

Dephosphorylation of pSB1A3

volume	ingredient
6µl	Antarctic phosphatase buffer
1µl	Antarctic phosphatase
3µl	dH2o

incubation at 37°C for 1 h

agarose- gel for digest/dephosphorylation

cut out correct bands
clean-up with qiagen-kit
concentrations:

pSB1A3	36,ng/µl
pIG15_002	4,4 ng/µl
pIG15_803-8	4,2 ng/µl

Ligation of self-build cloning site into pSBA3 (LS)

aim of this ligation is to get our constructs into pSB1A3, so that they can be expressed more easily
to distinguish between constructs in pSB1C3 and pSB1A3, those with the Amp- resistence will have an „A“ added after their name

	pIG15_803-A	pIG15_002-A	Ligation control
Insert	15µl	15µl	-
Vector	2µl	2µl	2µl
T4 Ligase	1µl	1µl	1µl
T4 Ligase buffer	2µl	2µl	2µl
dH2O	-	-	15µl

incubation at RT for 1h

Transformation of Ligation in E.coli T10 (LS)

transformation of ligation+ ligation control in E.coli Top 10
used 10µl of ligation (control) mix
incubation of LB-Amp plates with transformed E.coli cells o/n at 37°C

2015/05/25

Gibson for pIG15_403: checking plates (LS)

lots of clones on the plates
picked 8 clones
incubation at 37°C in LB-Clm for ~8h (shaking)

Ligation: checking plates (LS)

some clones on the ligation plates
nothing on the ligation control plate :)
picked 8 clones each (pIG15_803-8-A + pIG15_002-A)
incubation in LB-Amp at 37°C for ~8 h (shaking)

Miniprep: all clones from Gibson plate (pIG15_403) and ligation plates (LS)

2015/05/26

Test digest: HindIII + PstI

20µl digest, incubated at 37°C for 1h
agarose-gel: nothing but smears…

finally found out the reason: There was no RnaseA in buffer 1 of the biozym miniprep kit
miniprep will be repeated tomorrow, test digest as well

2015/05/27

Mini-Prep:

pIG15_403 (8x), pIG15_803-A (8x) and pIG15_002-A (8x) are prepped with the Biozym-Kit
according to the manufacturers protocol
eluted in 50 µl H2O

Nano-Drop

construct	concentration [ng/µl]	comment
pIG15_403 (1)	84.8
pIG15_403 (2)	71.9
pIG15_403 (3)	86.8
pIG15_403 (4)	118.1
pIG15_403 (5)	57.5
pIG15_403 (6)	80.4	–> sequencing (2015/05/29): correct
pIG15_403 (7)	96.7
pIG15_403 (8)	101.5
pIG15_803-A (1)	79.8
pIG15_803-A (2)	84.6
pIG15_803-A (3)	59.3
pIG15_803-A (4)	70.7
pIG15_803-A (5)	98.4
pIG15_803-A (6)	80.4
pIG15_803-A (7)	83.6
pIG15_803-A (8)	90.7	–> sequencing (2015/05/29): correct
pIG15_002-A (1)	108.8
pIG15_002-A (2)	74.5
pIG15_002-A (3)	101.7	–> sequencing (2015/05/29): correct
pIG15_002-A (4)	86.4
pIG15_002-A (5)	94.1
pIG15_002-A (6)	82.4
pIG15_002-A (7)	80.9
pIG15_002-A (8)	75.8

Test digest: BamHI + PstI

20µl digest (5µl Plasmid), incubated at 37°C for 1h

Transformation (RE) of pIG15_301 to pIG15_1101 in E. coli TOP10 according to the protocol

2015/05/28

Mini-Prep (RE)

yesterday's transformations
5 ml liquid culture (LB-amp)
Biozym-Kit
eluted in 30 µl dH2O

Nano-Drop:

Plasmid	concentration [ng/µl]
pIG15_301	212.2
pIG15_401	274.3
pIG15_501	222.1
pIG15_601	194.0
pIG15_701	215.9
pIG15_801	60.0
pIG15_901	220.9
pIG15_1001	220.7
pIG15_1101	220.0

2015/05/29

Digest (RE)

Plasmid	Enzyme 1	Enzyme 2
pIG15_301	BamHI	HindIII
pIG15_401	BamHI	HindIII
pIG15_501	BamHI	HindIII
pIG15_601	BamHI	HindIII
pIG15_701	BamHI	HindIII
pIG15_801	BamHI	HindIII
pIG15_901	BamHI	HindIII
pIG15_1001	BamHI	HindIII
pIG15_1101	BamHI	HindIII
pIG15_1701	BamHI	HindIII
pIG15_1801	BamHI	HindIII
pET22b+	BamHI	HindIII
pIG15_001-A	EcoRI	PstI
pIG15_403	EcoRI	PstI

incubation at 37°C for 1h
heat inactivation at 65°C for 15 min

gel electrophoresis and extraction (RE)

1% agarose gel
40 µl loaded
extraction with Qiagen-Kit
eluted in 30 µl dH2O

BamHI and HindIII digested antigens from pIG15_301 - pIG15_1001

* BamHI and HindIII digested antigens from pIG15_1101, pIG15_1701 and pIG15_1801; BamHI and HindIII digested pET22b+; EcoRI and PstI digested pIG15_001-A and pIG15_403

Plasmid	expected fragment size [bp]	extracted	concentration [ng/µl]
pIG15_301	1066	yes	6.6
pIG15_401	1066	yes	3.0
pIG15_501	1075	yes	3.4
pIG15_601	235	no	-
pIG15_701	1558	yes	20.9
pIG15_801	400	yes	4.6
pIG15_901	1237	yes	2.7
pIG15_1001	580	yes	2.0
pIG15_1101	1363	yes	4.4
pIG15_1701	547	yes	6.8
pIG15_1801	538	yes	8.4
pET22b+	5472	yes	2.4
pIG15_001-A	1441	yes	17.6
pIG15_001-A	~2000	yes	6.1
pIG15_403	2422	no	-
pIG15_403	2033	no	-

Ligation (RE)

pET22b+ (backbone) and 400 bp fragment from pIG15_801 (insert)

ingredient	amount [µl]	amount [µg]
backbone	15	36
insert	7.1	32.7
T4 Ligase	1
T4 Ligase Buffer	3
dH2O	4

incubation at RT for 1.5 h

Transformation (RE)

of the ligation product into E. coli TOP10 according to the protocol

2015/05/30

Mini-Prep (RE)

4 colonies from yesterday's transformation
5 ml liquid culture (LB-amp)
Biozym-Kit
eluted in 30 µl dH2O

Nano-Drop:

Plasmid	c [ng/µl]
pET22b+_801 (1)	575.8
pET22b+_801 (2)	247.7
pET22b+_801 (3)	309.9
pET22b+_801 (4)	260.0

Test-Digest (RE)

ingredient	amount [µl]
DNA	1
BamHI	1
HindIII	1
PstI	1
CutSmart	2
dH2O	14

incubation at 37°C for 1 h
heat inactivation at 65°C for 15 min
analysis on 1% agarose gel:

pET22b+_801 (pET22b+ incl Herpes simplex antigen) digested with BamHI, HindIII and PstI as well as undigested; expected fragment sizes: 4100, 1300 and 400 bp

2015/05/31

Mini-Prep (JN)

pET22b+_801 ligation product
same colonies as yesterday
Biozym-Kit

Nano-Drop:

Plasmid	Concentration [ng/µl]
pET22b+_801 - 1	142.6
pET22b+_801 - 2	38.9
pET22b+_801 - 3	66.7
pET22b+_801 - 4	106.0

Test-Digest (JN/RE)

~500 µg DNA
BamHI, HindIII and PstI (1 µl each)
incubation 1h at 37°C
analysis on 1% agarose gel:

pET22b+_801 digested with BamHI, HindIII and PstI as well as undigested plasmids; expected fragment sizes: 4100, 1300 and 400 bp

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Team:Freiburg/Labjournals/Cloning/May

Cloning

2015/05/01

2015/05/02

2015/05/03

2015/05/08

2015/05/10

2015/05/12

2015/05/12

2015/05/13

2015/05/14

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2015/05/19

2015/05/20

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2015/05/22

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2015/05/24

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2015/05/28

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2015/05/30

2015/05/31