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Cloning

2015/06/01

Primers (RE)

solved according to Sigma's instructions
1:10 dilution

Transformation

pET22b+ into E. coli TOP10

2015/06/02

Mini-Prep (RE)

5x 5 ml liquid culture (pEt22b+ in E. coli TOP10)
Biozym-Kit
eluted into 30 µl dH2O (1, 3, 5 into one tube; 2, 4 into another)

Nano-Drop: 109.6 ng/µl and 95.0 ng/µl

2015/06/03

PCR (LS/RE)

Salmonella antigens
2x 50 µl reactions for each antigen

Template	concentration [ng/µl]	fwd primer	rev primer
pIG15_1501	122	oIG15_1501f	oIG15_1502r
pIG15_1601	150	oIG15_1601f	oIG15_1602r

ingredient	amount [µl]
GC-Buffer	10 µl
fwd Primer	2.5 µl
rev Primer	2.5 µl
dNTP	1 µl
DMSO	1.5 µl
Phusion	0.5 µl
dH2O	32 µl
Template (~25 ng)	0.2 µl

Cycling conditions:

Step	Temperature [°C]	Duration [s]
Initial denaturation	92	180
Denaturation	92	20
Annealing	60	20
Extension	72	45
Final extension	72	600

PCR (LS/RE)

pET22b+ (109.6 ng/µl)
2x 50 µl reactions
fwd primer: pIG15_031f
rev primer: pIG15_030r

ingredient	amount [µl]
GC-Buffer	10 µl
fwd Primer	2.5 µl
rev Primer	2.5 µl
dNTP	1 µl
DMSO	1.5 µl
Phusion	0.5 µl
dH2O	32 µl
Template (~25 ng)	0.2 µl

Cycling conditions:

Step	Temperature [°C]	Duration [s]
Initial denaturation	92	180
Denaturation	92	20
Annealing	60	20
Extension	72	90
Final extension	72	600

repetition of the PCR (RE)

changes compared to before:

annealing temperature: gradient 55-72°C
denaturation: 98°C

2015/06/04

agarose gel with PCR from last night (Salmonella antigens 15 and 16 and pET igem Style) (LS)

cut out correct bands
clean up of PCR fragments with clean-up kit from Qiagen
concentrations:
Salmonella antigen 15
Salmonella antigen 16
pET22b+iGEM Style

Restriction digest with BamHI and HindIII (LS)

Salmonella antigen 15
Salmonella antigen 16
pIG15_1701 (1) (HIV)
pIG15_1801 (3) (Tpf1)
pIG15_601 (Rubella)

DNA	30µl
BamHI	1µl
HindIII	1µl
Cut Smart buffer	4µl
dH2O	4µl

incubation at 37°C for 1 h

PCR for pET22iGEM Style (RE)

will be filled in tomorrow

Agarose-gel for digest with BamHI and HIndIII (LS)

will be filled in tomorrow

2015/06/05

PCR (JN)

pIG15_1501 (122.9 ng/µl)
3x 50 µl reactions
fwd primer: pIG15_1501f
rev primer: pIG15_1502r

ingredient	amount [µl]
GC-Buffer	40 µl
fwd Primer	10 µl
rev Primer	10 µl
dNTP	4 µl
DMSO	6 µl
Phusion	2 µl
dH2O	127 µl
Template (~25 ng)	0.33 µl

Cycling conditions:

Step	Temperature [°C]	Duration [s]
Initial denaturation	98	180
Denaturation	98	20
Annealing	57	20
Extension	72	45
Final extension	72	600

25 cycles

Mini-Prep (RE)

pET22b+ iGEM style (Gibson)
pIG15_1701 and _1801 (Re-Trafo)
Biozym-Kit
eluted in 30 µl dH2O

Nano-Drop:

Plasmid	c [ng/µl]
pET22b+ iGEM style - 1	66.2
pET22b+ iGEM style - 2	59.1
pET22b+ iGEM style - 3	73.1
pET22b+ iGEM style - 4	67.0
pET22b+ iGEM style - 5	72.8
pET22b+ iGEM style - 6	40.7
pIG15_1701 - 1	178.8
pIG15_1702 - 2	212.1
pIG15_1801 - 1	167.1
pIG15_1801 - 2	156.0

Test-Digest (RE)

pET22b+ iGEM style constructs 1-6

ingredient	amount [µl]
DNA	8
PstI-HF	1
AflII	1
CutSmart	2
dH2O	8

–> incubation for 1 h at 37°C

analysis on 1% agarose gel:

PCR of antigen 15 (Salmonella). Expected fragment size: ~1700 bp. The amplified fragment is far too short.

Test-Digest of pET22b+ iGEM style (Gibson). Expected fragment sizes: ~4100 bp and ~1300 bp. Clone 1 shows the correct fragment sizes and will be sequenced

2015/06/06

PCR (RE)

amplification of antigen 8 (Herpes simplex) flanked with N-terminal 6xHis-tag and C-terminal Spy-tag

	[µl]
pIG15_801	0.5
oIG15_803f	10
oIG15_804r	10
Buffer	40
dNTP	4
DMSO	6
Phusion	2
dH2O	127.5

Cycling conditions:

	T [	C]
initial denaturation	98	180
denaturation	98	20
annealing	60	20
extension	72	20
final extention	72	600

Digest (RE)

pIG15_001-A with AflII and Acc65I to insert a double tagged antigen

	[µl]
Acc65I	1
AflII	1
Buffer 3.1	2
DNA	5
dH2O	11

2015/06/07

Miniprep (LS)

pETiGEM Style clones

pETiGEMStyle 7	58,1
pETiGEMStyle 8	71,1
pETiGEMStyle 9	66,6
pETiGEMStyle 10	62,8
pETiGEMStyle 11	64,1
pETiGEMStyle 12	24,2

Retrafo pIG15_001A (265,8ng/µl)

Digest (LS)

pETiGEM Style: PstIHf + AflII

10µl	DNA
1µl	PstIHf
1µl	AflII
2µl	Cut Smart Buffer
6µl	dH2O

pIG15_001A: Acc65I + AflII

5µl	DNA
1µl	Acc65I
1µl	AflII
2µl	Buffer 2.1
11µl	dH2O

incubation at 37°C for 1h
analysis on 1% agarose gel (RE)
no bands on any lane

PCR for Salmonella antigen 15 (RE)

pIG15_1501	0.2µl
oIG15_1501f	2.5µl
oIG15_1502r	2.5µl
Buffer	20µl
dNTPS	2µl
DMSO	3µl
Phusion	1µl
dH2O	64µl

–>2×50µl

cycling conditions

initial denaturation	98°C	180s
denaturation	98°C	20s
annealing	63°C	20s
extension	72°C	45s
final extension	72°C	10min
	4°C	infinity

–>analysis 1% agarose gel: successful amplification of ~1700bp fragment

2015/06/08

Mini-Prep (JN)

pET22b+ iGEM style (Gibson)
Biozym-Kit
eluted in 30 µl dH2O

Nano-Drop:

Plasmid	c [ng/µl]
pET22b+ iGEM style - 13	105.7
pET22b+ iGEM style - 14	111.7
pET22b+ iGEM style - 15	92.7
pET22b+ iGEM style - 16	123.8
pET22b+ iGEM style - 17	99.4
pET22b+ iGEM style - 18	91.3

Test-Digest (JN)

pET22b+ iGEM style constructs 13-18

ingredient	amount [µl]
DNA	5
PstI-HF	1
BamHI	1
CutSmart	2
dH2O	11

–> incubation for 1 h at 37°C

analysis on 1% agarose gel:

Gel-Ex (JN)

gel extraction of 1501 (Salmonella) PCR product from 2015/06/07
Qiagen Kit, elution with 25µl H2O
concentration: 8.6 ng/µl

PCR (JZ)

Salmonella antigen 15
according to the PCR protocol from 2015/06/07 (RE)

Transformation (JZ)

3 clones of pET22 iGEM style (13, 14 and 16) into E. coli TOP10

2015/06/09

Digest (RE)

Salmonella antigen 15 PCR products (unpurified)
enzymes were added directly to the PCR mix
BamHI, HindIII and DpnI –> 1 µl each
5 µl CutSmart buffer

analysis on 1% agarose gel (JN):

expected 1700 bp fragments on lane 1 and 2
excised to be extracted from the gel

PCR Salmonella antigen 15 (1700bp)

2015/06/10

Gel-Ex (RE)

Salmonella antigens (BamHI, HindIII and DpnI digested)
Qiagen-Kit
eluted in 20µl water (10min, 40°C, 300rpm)
concentration: 70,7 ng/µl

PCR for pET22iGEm Style (LS)

template: pET22b+ (109,7ng/µl)
Primer: oIG15_030f and oIG15_031r

buffer (high GC)	30µl
fwd primer	7,5µl
rev primer	7,5µl
dNTPs	3µl
DMSO	4,5µl
dH2O	95,3µl
Phusion polymerase	1,5µl
template	0,75µl

98°C	3min
98°C	20s
60°C	20s
72°C	2min
72°C	10 min
4°C	infinity

–> 25 cycles

PCR for pET22iGEMStyle did not work. Expected band length ~5500bp.

Repetition of the PCR for pET22 iGEM style (RE)

template: pET22b+ (95 ng/µl)
Primer: oIG15_030f and oIG15_031r

buffer (high GC)	30 µl
fwd primer	7.5 µl
rev primer	7.5 µl
dNTPs	3 µl
DMSO	4.5 µl
dH2O	92.5 µl
Phusion	1.5 µl
template	1.5 µl

–> 3x 50 µl

98°C	180 s
98°C	20 s
57°C	20 s
72°C	100 s
72°C	600 s
4°C	infinite

–> 25 cycles

Digest of the PCR products (RE)

5.5 µl CutSmart
1 µl DpnI
1 µl BamHI
directly added to the PCR mix
1 h at 37°C; 15 min at 65°C

1% agarose gel:

Expected fragment size ~5400 –> PCR did not work…

Repetition of the PCR for pET22 iGEM style (RE)

template: pET22b+ (95 ng/µl)
Primer: oIG15_030f and oIG15_031r

buffer	20 µl
fwd primer	5 µl
rev primer	5 µl
dNTPs	2 µl
DMSO	4 µl
dH2O	62 µl
Phusion	1 µl
template	0.8 µl

–> 2x 50 µl

98°C	180 s
98°C	30 s
63°C	20 s
72°C	240 s
72°C	600 s
4°C	infinite

–> 25 cycles

2015/06/11

Digest of the PCR products (RE)

5.5 µl CutSmart
1 µl DpnI
1 µl BamHI
directly added to the PCR mix
1 h at 37°C; 15 min at 65°C

2015/06/12

Gel-ex: pET22b+iGEMStyle in two fragments (LS)

excision of bands from agarose gel
clean-up with Qiagen kit
concentrations:
fragment 1: 8,7 ng/µl
fragment 2: 114,5 ng/µl

PCR for pET22b+iGEMStyle fragment 1 (RE)

Buffer	30µl
oIG15_030	7,5µl
pUC_ori	7,5µl
dNTPs	3µl
DMSO	4,5µl
Phusion polymerase	1,5µl
dH2O	96µl
pET22b+	0,75µl

3x 50 µl
cycling as above
annealing temperature: 56°C

Gel-ex for fragment 1 of pET22b+iGEMStyle (LS)

excision of fragment from agarose-gel
clean-uo with qiagen-kit
concentration: 109,5 ng/µl

Digest of fragment 1 and 2 with PciI and Acc65I (LS)

18µl	DNA
1µl	PciI
1µl	Acc65I
5µl	buffer 3.1
2,5µl	dH2O

incubation at 37°C for 1h
clean-up through agarose-gel/gel-ex wih qiage-kit
concentrations:
fragment 1: 18,1 ng/µl
fragment 2: 9,0 ng/µl

Ligation of fragment 1 and fragment 2 (LS)

3µl	fragment 1
18µl	fragment 2
2,5µl	T4 DNA ligase buffer
1µl	T4 DNA ligase
3µl	dH2O

incubation at 16°C o/n

2015/06/13

Transformation: Ligation of fragment 1 and 2 in E.coli T10 (LS)

10µl of ligation
transformtion according to protocol
plated on LB-Amp
incubation at 37°C

PCR (RE)

two fragments for pET22b iGEM style

template	fwd primer	rev primer
pET22b+	pUC_ori	oIG15_030r
pET22b+	oIG15_031f	pDrev4

Template	0.5 µl
fwd primer	5
rev primer	5
High GC Buffer	20
dNTPs	2
DMSO	3
Phusion pol.	1

–> 2 * 50 µl per fragment

cycling:

98°C	210 s
98	30
56	20
72	120
72	600
4	infinite

–> 20 cycles

Digest of the PCR products (RE)

1 µl DpnI
5.5 µl CutSmart Buffer
directly added into the PCR mix
1 h at 37°C; 15 min at 65°C

2015/06/14

Q5 PCR (RE)

two fragments for pET22b iGEM style
template and primers as for phusion PCR above

2x Q5 MaserMix	75 µl
fwd primer	7.5 µl
rev primer	7.5 µl
dH2O	59 µl
Template	1 µl

–> 3x 50 µl per fragment

Gel-Ex (RE)

Qiagen-Kit
two fragments for pET22b iGEM style
eluted in 20 µl dH2O

Nano-Drop:

fragment 1: 264 ng/µl
fragment 2: 305.8 ng/µl

2015/06/15

Test Digest of pET22b+_iGEMstyle1+2 (JN)

Expected fragment sizes ~1400bp and ~4100bp → did not work

Repetition of PCR for the two fragments of pET22b+iGEM Style (LS)

2xQ5 MM	98µl
fwd primer	9,8µl
rev primer	9,8µl
dH2O	77,3µl
pET22b+	1,8µl

4×50µl per fragment
cycling conditions as before
analysis on 1% agarose gel:
fragment one (two bands???)

upper band: 11,5ng/µl

lower band: 18,9ng/µl

fragment two:

62,6ng/µl

Digest (RE)

Q5 PCR products
5 µl NEB Buffer 3.1
1 µl PciI (Toolbox)
1 µl Acc65I
12 µl dH2O

agarose gel –> TOTAL DESTRUCTION…

2015/06/16

PCR for pET22b+iGEM Style as one fragment (LS)

Q5 polymerase

2xQ5MM	75µl
oIG15_030f	7,5µl
oIG15_031r	7,5µl
dH2O	59,4µl
pET22b+	0,6µl

98°C	3min
98°C	30s
56°C	20s
72°C	4min
72°C	10min
4°C	–

KOD polymerase

KOD MM	25µl
oIG15_030f	1,5µl
oIG15_031r	1,5µl
DMSO	2,5µl	–>only in one mix
dH2O	22/19,5µl

95°C	2min
95°C	20s
50°C	10s
70°C	2,5min
70°C	2,5min
4°C	–

–>30 cylcles each

analysis on 1%agarose gel
Q5 polymerase did not work
KOD polymerase did work!!!
concentrations:
KOD polymerase: 119,1ng/µl

Gel-Ex (RE)

KOD polymerase PCR product
Qiagen-Kit
eluted in 30 µl dH2O
Nano-Drop: 119.1 ng/µl

Digest (RE)

KOD polymerase PCR products

	1	2
DNA	7 µl	7 µl
CutSmart	5 µl	5 µl
dH2O	35 µl	36 µl
DpnI	1 µl	1 µl
BamHI	1 µl	-

Gel-Ex (RE)

digest products
fragment length ~5400 bp
Qiagen-Kit
eluted in 20 µl dH2O
DpnI digested: 12.1 ng/µl
DpnI/BamHI digested: 13.1 ng/µl

Ligation (RE)

9 µl pET22b (DpnI/BamHI digested)
2 µl T4 ligase buffer
1 µl T4 ligase
8 µl dH2O
16°C o/n

Gibson: fragment 1 and fragment 2 for pET22b+iGEMStyle (LS)

5µlDNA mix:

fragment 1	4,85µl
fragment 2	0,15µl

added 15µl Gibson mix
incubation at 50°C for 1h
transformation in E.coli T10

Gibson: 1fragment from KOD polymerase without Dpn1 digest (LS)

5µl of fragment after gel-ex
15µl of Gibson Mix
transformation in E.coli T10

2015/06/17

Transformation (RE)

Ligation product (10 µl)
1 fragment gibson

–> pET22 iGEM style

6 clones of pET22 iGEM style picked (1 fragment Gibson w/o DpnI digest (2015/07/16 LS))

Mini-Prep of 1-fragment Gibson for pET22b+iGEM Style without DpnI digest (LS)

mini-prep with Biozyem kit
concentrations:

pET22b+iGEMStyleG-1	22,8ng/µl
pET22b+iGEMStyleG-2	34,3ng/µl
pET22b+iGEMStyleG-4	40,8ng/µl
pET22b+iGEMStyleG-5	48,7ng/µl
pET22b+iGEMStyleG-6	53,3ng/µl

2015/06/18

Test-Digest (RE)

6 clones of pET22 iGEM style from yesterday
digested with PstI and AflII
expected fragment sizes: 1300 and 4100 bp

Mini-Prep (LS)

Ligation of pET22b+iGEMStyle
1-fragment Gibson of pET22b+iGEMStyle after DpnI digest
2-fragment Gibson of pET22b+iGEMStyle (fragment1 plus fragment 2)

	Ligation		1-fragment Gibson		2-fragment Gibson
1	81,4	1	91,8	1	92,1
2	78,9	2	71,2	2	59,1
3	67,0	3	46,9	3	77,2
4	104,3	4	120,8	4	141,6
5	75,9	5	70,8	5	108,7
6	81,6	6	72,2	6	82,4

Test-Digest (JN)

ingredient	amount [µl]	Master Mix x19 [µl]
DNA	5	-
PstI-HF	1	19
AflII	1	19
CutSmart	2	38
dH2O	11	209

–> incubation for 1 h at 37°C

analysis on 1% agarose gel:

expected fragment sizes ~1300bp and ~4100bp –> IT WORKED!!!

2015/06/19

Primer Annealing: oIG15_034f and oIG15_035r (LS)

5µM primer concentration: 10µl of 1:10 dilution of each primer plus 2µl NEB buffer 3
2,5µM primer concentration: 5µl of 1:10 dilution of each primer plus 2µl NEB buffer 3 plus 10µl dH2O
5 minutes at 95°C
slow cooling down (~1h)

Sequencing results pET22 iGEM (RE)

clone	primer	result
ligation 1	pBR3	correct
	pET-RP	mistake in lacI(?)
	pGL3-1	no seq. result
ligation 4	pBR3	correct
	pET-RP	correct
	pGL3-1	no seq. result
Gibson+ 1	pBR3	correct
	pET-RP	correct
	pGL3-1	no seq. result
Gibson+ 4	pBR3	correct
	pET-RP	correct
	pGL3-1	no seq. result

Trafo (RE)

pET22 iGEM Gibson+4 in E.coli TOP10

Digest (RE)

pET22 iGEM with BamHI and AflII
pIG15_803-A with BamHI and AflII
pET22b+ with BamHI and NotI

	pET22 iGEM	pIG15_803-A	pET22b+
DNA	9 µl (~1 µg)	20 µl (~2.5 µg)	10 µl (~1 µg)
Enzyme 1	1 µl	2 µl	1 µl
Enzyme 2	1 µl	2 µl	1 µl
CutSmart	5 µl	5 µl	5 µl
dH2O	34 µl	21 µl	33 µl

Expected fragment size of the backbone ~5400 bp can be seen

2015/06/20

Re-trafo (RE)

pET22b+
pIG15_001-A
pIG15_002-A
pIG15_803-A
pIG15_001
pIG15_002
pIG15_403
pIG15_803

PCR (RE)

804 –> Herpes simplex antigen with N-term. 6xHis-tag and C-term. Spy-tag

Buffer (GC)	30 µl
dNTPs	3 µl
DMSO	4 µl
Phusion	1.5 µl
dH2O	94.75 µl
Temp (pIG15_801)	1.25 µl
fwd (oIG15_803f)	7.5 µl
rev (oIG15_804r)	7.5 µl

–> 3x 50 µl

Cycling:

98°C	180 s
98°C	20 s
60°C	20 s
72°C	20 s
72°C	600 s
4°C	infinite

Primer dimers

repeated PCR (RE)

Buffer (GC)	24 µl
dNTPs	2.4 µl
DMSO	3.6 µl
Phusion	1.2 µl
dH2O	75.6 µl
Temp (pIG15_801)	1.2 µl
fwd (oIG15_803f)	6 µl
rev (oIG15_804r)	6 µl

–> 6x 20 µl

Cycling:

98°C	180 s
98°C	20 s
gradient: 55-72°C	20 s
72°C	20 s
72°C	600 s
4°C	infinite

Primer dimers

repeated PCR (RE)

Buffer (GC)	24 µl
dNTPs	2.4 µl
DMSO	3.6 µl
Phusion	1.2 µl
dH2O	91.6 µl
Temp (pIG15_801)	1.2 µl
fwd (oIG15_803f)	3 µl
rev (oIG15_804r)	3 µl

–> 6x 20 µl

Cycling:

98°C	180 s
98°C	20 s
gradient: 50-72°C	20 s
72°C	20 s
72°C	600 s
4°C	infinite

Primer dimers

2015/06/20

(JE) Digested the following antigens: pIG15_301

pIG15_401

pIG15_501

pIG15_601

pIG15_701

pIG15_801

pIG15_901

pIG15_1001

pIG15_1101

pIG15_1201

pIG15_1701

pIG15_1801

Digest with:

BamH1-HF	1 µl
Hind3-HF	1 µl
CutSmart	10 µl
DNA	5 µl (~1 µg)
ddH20	33 µl

(JE) Analysed the digest on 1% agarose gel:

Fragment sizes (bp) of the antigens:

pIG15_301	1066
pIG15_401	1066
pIG15_501	1075
pIG15_601	235
pIG15_701	1558
pIG15_801	400
pIG15_901	1237
pIG15_1001	580?
pIG15_1101	1363
pIG15_1201	3000?
pIG15_1701	547
pIG15_1801	538

Gel for digest of antigens 301-1201, 1701, 1801

(JE) According to the gel pic all bands but the 1001 and 1201 bands were extracted for a gel extraction. Conducted then a gel-extraction with the Qiagen-Kit. Eluted with 50 µl dH2O and 30 µl PE buffer (so 80 µl overall) (heard afterwards 20 µl is enough for extraction - blame me) Measured concentrations with the nanodrop (ng/µl):

pIG15_301	1066	3,4 ng/µl
pIG15_401	1066	2,7 ng/µl
pIG15_501	1075	5,4 ng/µl
pIG15_601	235	2,5 ng/µl
pIG15_701	1558	2,0 ng/µl
pIG15_801	400	3,5 ng/µl
pIG15_901	1237	3,7 ng/µl
pIG15_1001	580?	x
pIG15_1101	1363	3,5 ng/µl
pIG15_1201	3000?	x
pIG15_1701	547	3,1 ng/µl
pIG15_1801	538	3,1 ng/µl

Miniprep: pETiGEM (LS)

miniprep from plate
used biozym kit
concentration: 196,3ng/µl

pETiGEM liquid culture (LS)

inoculated 3x 5ml of LB-Amp
incubation at 37°C, shaking

Digest: pET22b+ and pETiGEM (LS)

pET22b+: BamHI HF and NotI HF
pETiGEM: BamHI HF and HindIII HF/ BamHI HF and AflII

2µl	Cut Smart buffer
28µl	dH2O
15µl	plasmid
1µl	each restriction enzyme

incubation at 37°C for 1h
analysis on 1%agarose gel

Digest did not work… Seems like the plasmids were completely cut into pieces.

Again Miniprep: pETiGEM (LS)

10ml with biozyme kit
concentrations: 83,4ng/µl and 519,5ng/µl

Repetition of digest: pET22b+ and pETiGEM (LS)

same digest as before
this time only 9µl of sample
used Philipp's enzymes instead of ours
two digest for each plasmid:

incubation for 30 min
incubation for 1h

digest worked this time!!! (gel pic was lost…)
Philipp did the digest as well, worked for him as well :)
picture is in the labbook (handwritten version)

2015/06/21

inoculation of yesterdays re-trafos (LS)

Kryo-Stock preparation

5ml LB
incubation at 37°C, shaking

Ligation (LS)

digested pET22b+ (Bam+Not)	1µl
5µM/2,5µM Primer Dimer Spy-Catcher-His	5µl
dH2O	12µl
T4 ligase buffer	2µl
T4 ligase	1µl

	antigen 1101	antigen1701
digested pEtiGEM (Bam+Hind)	1,8µl
digested antigen	8,06µl	2,21µl
dH2O	8µl	14µl
T4 ligase buffer	2µl
T4 ligase	1µl

incubation at 16°C, o/n

Repetition of PCR for fragment 804 (LS)

Fragment 804 = antigen 8 (HSV 1) with N-terminal His-Tag and C-terminal Spy-Tag

Q5 polymerase		Dream Taq
Q5 buffer	20µl	DreamTaq buffer	20µl
dNTPs	2µl	dNTPa	2µl
oIG15_803f	2,5µl	oIG15_803f	2,5µl
oIG15_804r	2,5µl	oIG15_804r	2,5µl
high GC buffer	20µl
Q5 polymerase	1µl	Dream Taq	1µl
dH2O	32µl	dH2O	53µl

Q5 polymerase		Dream Taq
98°C	3min	95°C	30s
98°C	30s	95°C	20s
50°C-70°C	20s	45°C-60°C	50s
72°C	20s	68°C	1min
72°C	2min	68°C	5min
4°C

DMSO only in every second tube
analysis on 1% agarose gel: PCR did not work with Q5 polymerase, but it did work with Dream Taq!!!

Mini-Prep of yesterday's re-trafo (LS)

biozym kit

concentrations
pIG15_002	64,2
pIG15_403	66,4
pET22b+	61,0
pIG15_803-A	50,2
pIG15_001	69,9
pIG15_803	94,3
pIG15_001-A	108,6
pIG15_002-A	181,4

prepared Gly-stocks of all plasmids
300µl 50% glycerol
500µl liquid culture

2015/06/22

Inoculation of ligation clones (LS)

We need to express the Spy-Catcher, so that it can be used by the SurfaceChemistry Group to work on Spy-Catcher-Spy-Tag interaction. For purification we added a His-tag that is introduced via the primers.

3x pET22b+ with Spy-Catcher-His
4x pETiGEM + antigen 1101
4x pETiGEM + antigen 1701

Digest (LS)

	pIG15_803	pETiGEM	fragment 804
plasmid	10µl	2µl	5µl
BamHI Hf	1µl	–
AflII	1µl	1µl	1µl
Cut Smart buffer	5µl	5µl	5µl
dH2O	33µl	42µl	39µl

incubation at 37°C
digest of fragment 804 worked
nothing for pETiGEM and only backbone for pIG15_803

Digest of pIG15_803, pETiGEM and fragment 804

Repetition of digest (LS)

same as above
only pETiGEM and pIG15_803
again: nothing for pETiGEm and only backbon for pIG15_803

Digest: pETiGEM and pIG15_803-a (RE)

Trafo: pETigem in E.coli T10 (RE)

2015/06/23

Miniprep: pETigem (LS)

miniprep with peqlab kit
concentrations: ? and ?

Repetition of digest of pETiGEM with AflII

Miniprep: ligation clones (antigen 11 and 17 in pETiGEM without a tag)

biozyme kit
concentrations:

pETigem_1102 (1)	58,4
pETigem_1102 (2)	315,7
pETigem_1102 (3)	126,6
pETigem_1102 (4)	163,4

pETigem_1702 (1)	139,4
pETigem_1702 (2)	121,7
pETigem_1702 (3)	436,4
pETigem_1702 (4)	101,1

pET22b+ Spy-Catcher with His-tag (1)	85,8
pET22b+ Spy-Catcher with His-tag (3)	85,5

Test digest: antigens 11 and 17 in pETiGEM and pET22b+Spy-Catcher with His-tag (LS)

5µl	plasmid
1µl	re 1
1µl	re 2
2µl	Cut Smart
11µl	dH2O

pETiGEM_1102/1702 with PtsIHF and BamHI HF
pET22b+Spy-Catcher with His-tag with PstIHF and NotHF

Test digest for pETiGEM with antigen 11 (CT) or 17 (HIV) [upper part] and pEt22b+Spy-Catcher with His-tag [lower part]

2015/06/24

gel-ex of digested fragment 804 (AflII) and digested pETigem (AflII) (LS)

concentrations:
fragment 804: 14,5 ng/µl
pETiGEM: 17,3ng/µl

digest: fragment 804 (HSV1 antigen with C-term Spy-Tag and N-term His-Tag) and pETiGEM with Acc65I (LS)

15µl	DNA
5µl	buffer 3.1
1µl	Acc65I
29µl	dH2O

repetition of ligation: pET22b+ and Spy-Catcher with His-tag (LS)

same way as before (see 2015/06/20) + ligation control
this time ligation on RT for 1h

transformation of ligation in E.coli Top 10 (LS)

10µl of ligation
transformation according to protocol
incubation on LB-Amp plates at 37°C

gel-ex of digested fragmet 804 (HSV1 antigen with C-term Spy-tag and N-term His-tag) and pETiGEM (Acc65I) (LS)

concentrations:
fragment 804: 5,3 ng/µl
pETigem: 4,8 ng/µl

Ligation of fragment 804 (HSV1 antigen with C-term Spy-tag and N-term His-tag) and pETiGEM (RE)

11µl	pETiGEM
4µl	fragment 804
2µl	T4 ligase buffer
1µl	T4 ligase

2015/06/25

Transformation of ligation (RE)

10µl of the ligation
transformation in E.coli T10

pET22b+Spy-Catcher-His (RE)

picked 4 clones from each plate (2,5µM/5µM)
incubation in 5ml LB-Amp at 37°C, shaking

2015/06/26

Miniprep: pETiGEM_804 (JN)

biozyme kit
concentrations:

pETigem_804 (1)	197.3
pETigem_804 (2)	85.3
pETigem_804 (3)	67.6
pETigem_804 (4)	78.8

2015/06/27

Test-digest of pET22b+ with Spy-Catcher with His-tag, pETigem AflII, pETigem (JN)

To verify integration of the Spy-Catcher into the pET22b+ a test digest was performed as follows. At the same time pETiGEM was digested with AflII and BamHI, for ligation with fragment 803 (=antigen HSV1 with C-term His-tag). One already AflII digested pETiGEM was only digested with BamHI.

pET22b+ with Spy-Catcher: PstI-HF and XhoI-HF
pETigem AflII: BamHI-HF
pETigem: AflII and BamHI-HF

ingredient	pET22b+ with Spy-Catcher	pETigem AflII	pETigem
DNA	5µl	18µl	10µl
enzyme1	1µl	1µl	1µl
enzyme2	1µl	-	1µl
CutSmart	5µl	5µl	5µl
dH2O	38µl	26µl	33µl

incubation 1h at 37°C
dephosphorylation of both pETigem plasmids to avoid re-ligation of the backbone

Digest	50µl
Antarctic Phosphatase buffer	6µl
Antarctic Phosphatase	1µl
dH2O	3µl

incubation 1h at 37°C
analysis of all digests on 1% agarose gel

Gel-Ex of pETigem (JN)

concentration: 12.0ng/µl

Ligation of pETigem and 803 (HSV1 with C-term His-tag) (JN)

ingredient	pETigem+803	pETigem control
pETigem	4.2µl	4.2µl
803	3.8µl	-
ligase buffer	2µl	2µl
T4 ligase	1µl	1µl
dH2O	9µl	12.8µl

* overnight at 16°C

2015/06/28

Trafo (JN)

trafo of pETigem_803 and pETigem ligation control (=just backbone without 803-insert)

2015/06/29

Test-digest of pET22b+ with Spy-Catcher with PstI-HF and EcoRI-HF (JN)

pET22b+Spy-Catcher (2.5µM) 1-4
pET22b+Spy-Catcher(5µM) 1-4
pET22b+ digested and undigested (as control)

ingredient	pET22b+Spy-Catcher	pET22b+ digested	pET22b+ undigested
DNA	5µl	5µl	5µl
PstI-HF	1µl	1µl	-
EcoRI-HF	1µl	1µl	-
CutSmart	2µl	2µl	2µl
dH2O	11µl	11µl	13µl

20µl sample
analysis on 1% agarose gel:

expected fragment size for pET22b+Spy-Catcher with His-tag ~5500bp, pET22b+ digested should be two fragments → correct!

expected fragment size for pET22b+Spy-Catcher with His-tag ~5500bp → correct!, fourth lane is somehow missing

the lower band of pET22b+Spy-Catcher (5µM) (1) could be due to only one Spy-Catcher-fragment in the plasmid, the other ones could have more than one Spy-Catcher-Fragment inserted.
pET22b+Spy-Catcher (5µM) (1) and (2), pET22b+Spy-Catcher (2.5µM) (1) sent in for sequencing

Mini-Prep (RE)

8 clones of pET_803
Qiagen-Kit
eluted in 30 µl dH2O

Nano-Drop:

pET_803 - 1	205.1 ng/µl
pET_803 - 2	200.5
pET_803 - 3	149.5
pET_803 - 4	215.7
pET_803 - 5	227.1
pET_803 - 6	229.9
pET_803 - 7	242.5
pET_803 - 8	212.1

Test-Digest (RE)

pET_803 - 1-8
Fermentas FastDigest
1 µl BamHI
1 µl HindIII
2 µl FD Buffer
5 µl DNA
11 µl dH2O
(a little more than) 5 min at 37°C

analysis on 1% agarose gel:

TestDigest of pET_803 (Herpes simplex antigen with 10xHis-tag in pET_iGEM) with BamHI and HindIII (Fermentas): expected fragment sizes ~ 800 and ~ 5400 bp –> larger fragment is not visible, but clones 1 and 2 will be sequenced anyway

Trafo (RE)

BioBricks for Interlab study:

BioBrick	resistance	plate	well
Bba_K823005	Cml	1	20K
Bba_K823008	Cml	1	22A
Bba_K823013	Cml	1	22K
Bba_I13504	Cml	3	17 C

–> solved in 10 µl dH2O –> 2 µl (~ 500 pg) used for transformation

pET22b+ (Re-trafo)

2015/06/30

TestDigest of pET_804 (RE)

4 clones
PstI-HF and HindIII-HF (1 µl each)
2 µl CutSmart
5 µl DNA
11 µl dH2O

analysis on 1% agarose gel:

[

Taq PCR for fragment 804 (RE)

804 = Herpes simplex with N-term. 6xHis- and C-term. Spy-tag

Dream Taq Buffer	37.5 µl
dNTPs	3.75 µl
DMSO	4.5 µl
oIG15_803f	7.5 µl
oIG15_804r	7.5 µl
pIG15_801	7.5 µl
Dream Taq polymerase	3 µl
dH2O	78.75 µl

–> 3 x 50 µl

Cycling:

95°C	30 s
95°C	20 s
55°C	50 s
68°C	60 s
68°C	300 s
4°C	infinite

analysis on 1% agarose gel:

PCR product 804: expected fragment size ~800 bp –> referring band was exciced

Gel-Ex (RE)

PCR product 804
all lanes were loaded on the same column
Qiagen-Kit
eluted in 30µl dH2O
Nano-Drop: 241 ng/µl

Digest of fragment 804 and pET-iGEM(RE)

804	pET_iGEM
DNA	6 µl (1.5 µg)	10 µl (1.3 µg)
Acc65I	1 µl	1 µl
NEB Buffer 3.1	5 µl	5 µl
dH2O	38 µl	34 µl

–> 1h at 37°C

analysis on 1% agarose gel:

Gel-Ex (RE)

fragment 804 and pET_iGEM (Acc65I digested)
Qiagen-Kit
eluted in 20 µl dH2O

Nano-Drop: - 804: 23 ng/µl - pET_iGEM: 4 ng/µl

Digest of fragment 804 and pET_iGEM (RE)

FastDigest (Fermentas)
1 µl BspTI and 2 µl FD Green Buffer added to the Gel-Ex elution
5 min at 37°C
5 min at 80°C (heat inactivation)
cool down to RT

Ligation of fragment 804 and pET_iGEM (RE)

without cleaning up the digest products
both digest products were pooled
1 µl T4 Ligase and 5 µl T4 Liagse Buffer were added
1 h at RT

Team:Freiburg/Labjournals/Cloning/June

Cloning

2015/06/01

2015/06/02

2015/06/03

2015/06/04

2015/06/05

2015/06/06

2015/06/07

2015/06/08

2015/06/09

2015/06/10

2015/06/11

2015/06/12

2015/06/13

2015/06/14

2015/06/15

2015/06/16

2015/06/17

2015/06/18

2015/06/19

2015/06/20

2015/06/20

2015/06/21

2015/06/22

2015/06/23

2015/06/24

2015/06/25

2015/06/26

2015/06/27

2015/06/28

2015/06/29

2015/06/30