Team:Freiburg/Notebook/Cloning

From 2014.igem.org

The AcCELLerator

Cloning

The protocols for our methods can be found here.

During our experiments, we conducted a lot of PCRs with different Primers. In order to stay on top of things, we numbered Primers, PCRs and constructs. You can download both lists with detailed information about Primer composition and about PCRs in the following documents:


p14rz_002

  • PCR: o14rz_005, o14_rz006; Template pQXCIN-SLC7A1
  • Digest: pKM006, EcoRI + NotI; PCR-product
  • Testdigest with PstI in Cutsmart, expected bands: 5,9; 0,7

PCR

Testdigest

p14rz_003

  • PCR 3
  • Digest 3, 4
  • Testdigest with PstI + SpeI in Cutsmart, expected bands: 4,1;2,1;1,8

PCR

Digest

Testdigest

p14rz_004

  • PCR 21,22
  • Digest -
  • Testdigest with PstI in Cutsmart, expected bands: 4,1;3,2

PCR

PCR

Testdigest

p14rz_005

  • PCR 4,5
  • Digest 5
  • Testdigest with AgeI+HindIII in Cutsmart, expected bands: 6,6;1,3

PCR

Digest

Testdigest

p14rz_006

  • PCR 17,18
  • Digest 5
  • Testdigest with AgeI+HindIII in Cutsmart, expected bands: 6,6;1,4

PCR

Digest

Testdigest

p14rz_007

  • PCR 37,38
  • Digest 21
  • Testdigest with PstI in Cutsmart, expected bands: 4,1;2,2;1,7

PCR

Digest

Testdigest

p14rz_008

  • PCR blue light receptor; Template: pQXCIN-SLC7A1
  • Digest pKM006 EcoRI, NotI
  • Testdigest with SmaI in Cutsmart, expected bands: 5,7

PCR

Testdigest

p14rz_009

  • PCR 39,40
  • Digest 5
  • Testdigest with BamHI in Cutsmart, expected bands: 3,0;2,7

Digest

Testdigest

p14rz_010

  • PCR 23,24
  • Digest 12
  • Testdigest with PstI in Cutsmart, expected bands:3,1;2,3;0,56

PCR

PCR

Testdigest

p14ls_003

  • PCR 7,9,10
  • Digest -
  • Testdigest with KpnI in Cutsmart, expected bands: 4,9; 1,3; 0,4

PCR

Digest

Testdigest

p14vv_001

  • PCR Primers vv003,vv004; Template: pMIG
  • Digest pMIG EcoRI,NotI; C1-eGFP EcoRI,NotI
  • Testdigest with NgoMIV in Cutsmart, expected bands: 5,7; 0,6

Digest

Digest

Testdigest

p14vv_002

  • PCR -
  • Digest 8,11
  • Testdigest with PstI in Cutsmart, expected bands: 3,2;2,1;1,4

PCR

Digest

Testdigest

p14vv_003

  • PCR pMIG with primer vv003+vv004-
  • Digest xx
  • Testdigest with KpnI in Cutsmart, expected bands: 3,5;1,5

PCR

Testdigest

p14vv_006

  • PCR 26
  • Digest 13
  • Testdigest with NgoMIV in Cutsmart, expected bands: 3,5;1,9;0,95,0,6

PCR

Testdigest

p14vv_007

  • PCR 27
  • Digest 14
  • Testdigest with NgoMIV in Cutsmart, expected bands: 3,5; 1,3; 0,97; 0,66

PCR

Testdigest

p14vv_008

  • PCR 43
  • Digest 22
  • Testdigest with EcoRV in Cutsmart, expected bands: 3,4; 1,4 0,75

PCR

Digest

Testdigest

p14vv_009

  • PCR 44
  • Digest 22
  • Testdigest with EcoRV+MluI in Cutsmart, expected bands: 3,4; 1,43; 0,75

PCR

Digest

Testdigest

p14vv_010

  • PCR 45
  • Digest 22
  • Testdigest with EcoRV in Cutsmart, expected bands: 3,4; 2,2

PCR

Digest

Testdigest

p14vv_012

  • PCR 46
  • Digest 22
  • Testdigest with EcoRV in Cutsmart, expected bands: 4,7;3,4

PCR

Digest

Testdigest

p14vv_013

  • PCR 47,48,49
  • Digest 22
  • Testdigest with NgoMIV in Cutsmart, expected bands: 3,4;2,0;1,3;0,2

PCR

Digest

Testdigest

p14vv_014

  • PCR 50,51
  • Digest 22
  • Testdigest with NgoMIV in Cutsmart, expected bands: 4,1; 1,56; 0,66

PCR

Digest

Testdigest

p14zl_010

  • PCR 1,2
  • Digest 1
  • Testdigest with NgOMIV in Cutsmart, expected bands: 4,7;2,4;1,1

PCR

Digest

Testdigest

p14zl_011

  • PCR 19,20
  • Digest 1
  • Testdigest with NgOMIV in Cutsmart, expected bands: 4,7;2,0;1,3;0,43

PCR

Digest

Testdigest

p14zl_016

  • PCR 52,53
  • Digest 22
  • Testdigest with HindIII in Cutsmart, expected bands: 5,7;2,3;1,7

PCR

Digest

Testdigest

p14zl_017

  • PCR 64,65
  • Digest 24
  • testdigest with NgoMIV in Cutsmart, expected bands: 5,4;2,9

PCR_digest

Testdigest

p14zl_020

  • PCR 59,60
  • Digest 24
  • testdigest with BamHI-HF, NgoMIVin Cutsmart, expected bands: 4,7; 2,4; 1,8

PCR

Digest

Testdigest

Standardization

Standardization - August

PCR mix for site-directed mutagenisis (25 µl)

template1 µl
buffer (5x)5 µl
Primer1 µl each
polymerase0.5 µL
dNTPs1 µl
DMSO0.5 µl
water15 µl

start your PCR mix with just one primer and let reaction run for ten cycles including end elongation. shortly after that add 1 µl dNTP and 1 µl of primer 2. start second PCR under same conditions for 15 cycles. Don't spend too much time with adding dNTPs and primer 2.

DNA amplifications such for ligation PCR approaches contained 50 µl.

protocoll site-directed mutagenisis

98 °C 5 min denaturation
98 °C 30 s denaturation
60 °C 30 s annealing
72 °C 30 s/kb elongation
72 °C 10 min end elongation

To avoid high background activities by not-mutated plasmids, the PCR mixture was digested by DpnI. It cuts methylated DNA only, leading to remove the DNA template.

Therefore transformation is done with unmethylated PCR product, where plasmids contain the desired mutation.

DpnI digest

add 3 µl Cutsmart, 0.5 µl water and 0.5 µl DpnI. Incubate at 37 °C for 90 minutes, followed by an inactivation step at 80 °C for 20 minutes.

test digest

2 µl bufferabout 500 - 1000 ng DNA0.5 µl enzymeup to 20 µl water

PCR products or test digests of plasmids where separated gelelectrophoresis, containing 1 % agarose. Small gels were run at 95 V for 90 minutes, large gels at 105 V for 90 minutes.

2014.08.21.


PCR #templateprimer 1primer 2polymeraseproductsizeannealing temperatureelongation time
1pKM297o14_sd_003o14_sd_004Q5ePDZb_G1448C_PstI3.8 kb60 °C01:55 min
2pKM292o14_sd_011o14_sd_012Q5GAL4_binding_domain_RFC_250.5 kb60 °C00:16 min
3pKM292o14_sd_019o14_sd_020Q5LOV_C3423T_PstI3.7 kb6001:53 min
4zl_003o14_sd_023o14_sd_024Q5P2A_RFC_100.1 kb6000:04 min
5zl_003o14_sd_027o14_sd_028Q5P2A_C9374G_NgoMIV11.7 kb6005:50 min

Each PCR was done in triplicates and digested with DpnI. Each replicate was used for transformation with competent bacteria on a seperated Ampicillin plate and incubated at 37 °C. bacteria containing zl_003 were incubated at 32 °C.

2014.08.22.


PCR #templateprimer 1primer 2polymeraseproductsizeannealing temperatureelongation time
6zl_oo3o14_sd_029o14_sd_030Phusionpuromycin_resistance_gene_RFC_250.6 kb60 °C00:20 min
7rz_008o14_sd_008o14_sd_009PhusionCAT-1_C1168T_PstI6.4 kb60 °C03:15 min
8pKM084o14_sd_047o14_sd_048PhusionSEAP_G1419C_PstI5.4 kb6002:45 min

All PCRs were digested with DpnI and transformed on Ampicillin containing plates at 37 °C.

Transformation from PCR 1.1/1.2/1.3, 3.1/3.2/3.3, 5.1/5.3 were successful, but entirely PCR 2 & 4 not (repeated with inkubation at 32 °C). Three colonies were picked from each plate and used for over night culures (ONC).

Advice from instructor: an elongation time of 30 s for each DNA fragment smaller than 1 kb will be used.

2014.08.23.

A mini prep kit from Quiagen (250 reactions) were used for DNA isolation:

templateconcentration [ng/µl]templateconcentration [ng/µl]
1.1 I155.2 ng/µl1.2 I174.0 ng/µl
1.1 II153.1 ng/µl1.2 II727.5 ng/µl
1.1 III566.6 ng/µl1.2 III562.5 ng/µl
1.3 I265.8 ng/µl3.1 I174.1 ng/µl
1.3 II226.4 ng/µl3.1 II144.5 ng/µl
1.3 III187.7 ng/µl3.1 III144.4 ng/µl
3.2 I197.0 ng/µl3.3 I163.1 ng/µl
3.3 II163.5 ng/µl5.1 I150.0 ng/µl
5.3 I448.7 ng/µl5.1 II442.2 ng/µl
5.3 II230.1 ng/µl5.1 III295.7 ng/µl
5.3 III559.4 ng/µl

test digest with:

PCRenzymebufferfragment size
1PstI/ClaICutSmartmutated: 2.4 kb, 1.5 kb, not mutated: 2.4 kb, 0.9 kb, 0.6 kb
3PstI/BbsICutSmartmutated: 2.4 kb, 0.65 kb, 0.53 kb; not mutated: 2.4 kb, 0.53 kb, .04 kb, .026 kb, 0.23 kb, 0.06 kb
5AgeI/NgoMIVNEB 1.1mutated: 5.1 kb, 4.7 kb, 1.8 kb; not mutated: 4.7 kb, 4.1 kb, 2.8 kb

0.625 µl BbsI was added, due to its decreased activity (75%) in NEB 1.1. Test digests were done over night.

Transformations of PCR 8 and PCR 5 didn't work. Repeated in duplicates: SEAP was incubated at 37 °C, P2A at 32 °C.

5 ONC of PCR 7 (CAT-1) were done.

2014.08.24.

results of gel electrophoresis:

Description of Image

gel electrophoresis of PCR 1 & 5.

PCR 1:         PCR 1.1 I and PCR 1.1 II showed perfect fragment sizes (2.3 kb & 1.4 kb). àCandidates for sequencing.

PCR 5:         PCR 5.1 & PCR 5.3 were successful. DNA fragments at 5.1 kb and 4.7 kb. Fragment at 2 kb invisible, but if DNA wasn’t mutated, there would not be a fragment at 5.1 kb. à Candidates for sequencing.

Description of Image

gel electrophoresis of PCR 3.

PCR 3:         PCR 3.1 III looked fine. Fragments as expected.à Candidate for sequencing.

Despite missing sequencing results, PCR 5.1 I was used for a transformation.

DNA extraction of CAT-1 (5x)

PCR #

Concentration [ng/µl]

7 I

489.2

7 II

316.5

7 III

385.0

7 IV

451.8

7 V

550.0

 

Test digest with:

PCR #

enzyme

buffer

fragment size

7

AgeI/PstI-HF

CutSmart

mutated:4.2 kb, 1.6 kb

not mutated: 4.2 kb, 1.3 kb, 0.3 kb

test digest was done overnight.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

10

pKM292

o14sd_011

014sd_012

Phusion

GAL4_RFC_25

0.5 kb

55 °C

11

zl_003

o14sd_29

014sd_030

Phusion

Puromycin_resistance_gene_RFC_25

0.6 kb

55 °C

 

2014.08.25

 

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

12

pKM084

o14sd_047

014sd_048

Phusion

SEAP_G1419C_PstI

5.4 kb

55 °C

13

zl_003

o14sd_055

014sd_056

Phusion

WPRE_C530A_NgoMIV

11.7 kb

55 °C

14

LOV PCR Product

o14sd_011

014sd_012

Phusion

GAL4_ RFC_25

0.5 kb

55 °C

 

Result gel electrophoresis (digest CAT-1)

Description of Image

gel electrophoresis of PCR 7.

Result of colony 1 looked fine. à Candidate for sequencing.

Transformation of PCR 6 & 8 did not work.

2014.08.26

DNA extraction from PCR 5.1 (5x)

PCR #

Concentration [ng/µl]

5.1 I

213.0

5.1 II

283.2

5.1 III

254.1

5.1 IV

198.4

5.1 V

232.2

 

Test digests according to 2014.08.23 overnight.

2014.08.27

Gel electrophoresis from test digest PCR 5.1

Description of Image

gel electrophoresis of PCR 5.1.

Test digests looked fine. Fragments as expected at 5.1 kb, 4.7 kb and about 2.0 kb. PCR 5.1 I was used as a template to amplify P2A with RFC 25 prefix and suffix.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

15

PCR 5.1 I

o14sd_025

014sd_026

Phusion

P2A_RFC_25

0.1 kb

55 °C

PCR 15 was done in 50 µl approach and triplicates.

Gel electrophoresis from PCR 15

Description of Image

gel electrophoresis of PCR 15.

Gel contains 2 % agarose. Marker is 100 bp from Promega. Fragments lay between 100 and 200 bp. Theoretical size 136 bp. àFragments cut out and prepared for gel extraction. All three fragments were run over one column.

PCR 14 repeated as PCR 16 due to contaminated water.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

16

LOV PCR Product

o14sd_011

014sd_012

Phusion

GAL4_ RFC_25

0.5 kb

55 °C

 

Gel electrophoresis from PCR 16

Description of Image

gel electrophoresis of PCR 16.

Gel contains 2 % agarose. Marker is 100 bp from Promega. Fragments lay at 500 bp, expected size: 517 bp. Fragments were cut out and prepared for gel extraction. All three fragments were run over one column.

Gel extraction from P2A RFC 25 and GAL4 RFC 25

name

concentration [ng/µl]

GAL4 RFC 25

38.5

P2A RFC 25

34.4

 

Ligation of P2A RFC 25 and GAL4 RFC 25 in pSB1C3

Preparative digest according to test digest protocol, but incubated for four hours. Inserts and backbone were cut with EcoRI-HF/PstI-HF in CutSmart.

Used ratio backbone:insert = 1:3

Used volumes of insert and backbone

GAL4 RFC 25

1.72 µl

pSB1C3

0.48 µl

P2A RFC 25

1.7 µl

pSB1C3

0.5 µl

control (water)

1.7 µl

pSB1C3

0.5 µl

Ligation was performed with T4 ligase (40.000 U/ml) from NEB

After transformed with DNA bacteria culture were incubated at 37 °C for one hour. Afterwards cultures were streaked out onto agar plates containing Chloramphenicol.

Despite missing sequencing results, ONC were made from PCR 1.1 I & PCR 3.1 III (5x each).

2014.08.28

Sequencing results: P2A not mutated, CAT-1: fail. nucleotide length 1 nt., LOV: not been uploaded.

DNA extraction from ONC PCR 1.1 I (ePDZb) & PCR 3.1 III (LOV)

template

concentration [ng/µl]

template

concentration [ng/µl]

LOV I

103.7

ePDZb I

136.8

LOV II

91.4

ePDZb II

121.4

LOV III

119.4

ePDZb II

137.0

LOV IV

113.4

ePDZb IV

102.2

LOV V

129.9

ePDZb V

155.2

 

Test digests PCR 1.1 I & PCR 3.1 III:

template

enzyme

buffer

fragment size

LOV

XhoI/PstI-HF

CutSmart

mutated:2.7 kb, 0.7 kb

not mutated: 2.7 kb, 0.4 kb, 0.3 kb

ePDZb

ClaI/PstI-HF

CutSmart

mutated:2.3 kb, 1.5 kb

not mutated: 2.3 kb, 0.9 kb, 0.6 kb

 

Description of Image

gel electrophoresis of LOV & ePDZb.

LOV didn’t work -> no fragment at 0.7 kb. ePDZb looked fine. Visible fragment at 1.5 kb as expected.

Ligation of P2A RFC 25 and GAL4 RFC 25 didn’t work. Next time preparative digest will be desalted by PCR purification kit.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

17

zl_003

o14sd_023

014sd_024

Q5

P2A_RFC_10

0.1 kb

55 °C

18

zl_003

o14sd_057

014sd_058

Q5

WPRE_RFC_10

0.6 kb

55 °C

19

PCR 5.1 II

o14sd_039

014sd_040

Q5

CAT-1_RFC_10

1.9 kb

50 °C

20

ePDZb I

o14sd_005

014sd_006

Q5

ePDZb_A1775G_EcoRI

3.8 kb

50 °C

21

pKM084

o14sd_047

014sd_048

Q5

SEAP_G1419C_PstI

5.4 kb

50 °C

PCR 20 & 21 digested by DpnI and afterwards transformed onto Ampicillin plates and incubates at 37 °C

PCR 17 -19 were directly purified preparedly digested overnight with EcoRI-HF/PstI-HF in Cutsmart.

Apporach:          28 µl eluate

                               1 µl enzyme each

                               4 µl CutSmart

                               6 µl water

2014.08.30

ONC from GAL4 RFC 25 in pSB1C3

A Chloramphenicol plate from GAL4 RFC 25 ligation (2014.08.27.) was found in the 37 °C incubator. One colony was visible. à Three ONC were picked from this colony.

ONC from PCR 20 & 21

Description of Image

gel electrophoresis of PCR 17 & 18.

Plates were settled with bacteria. Three ONC were picked from each plate.

P2A looked fine. The expected size was 134 bp. Fragments were above the100 bp lane and cut out for gel extraction. WPRE showed expected lane sizes (646 bp.). Cut out for gel extraction.

Description of Image

gel electrophoresis of PCR 19.

Instead of 2-logfrom NEB, Promega’s 100 bp ladder was used. Expected size for CAT-1 RFC 10 were 1577 bp. The highest lane of DNA ladder is 1.5 kb -> PCR products were cut out for gel extraction.

All triplicates of one sample were run over one column.

Gel extraction from preparative digests (2014.08.29.)

name

concentration [ng/µl]

P2A RFC 10 (PCR 17)

22.9

WPRE RFC 10 (PCR 18)

53.9

CAT-1 RFC 10 (PCR 19)

17.4

 

Ligation of WPRE RFC 10, CAT-1 RFC 10, P2A RFC 10 in pSB1C3

ratio backbone:insert = 1:3

 

Volume [µl]

Volume backbone [µl]

P2A

1.68

0.52

CAT-1

1.64

0.56

WPRE

1.83

0.37

control (water)

1.72

0.48

 

We got a new ligation buffer from our instructor à Quick ligation buffer. Used buffer volume: 2.6 µl, 2.4 ligation volume and 0.2 T4 ligase (400.000 U/ml) from NEB.

5 µl ligation approaches were used for transformation. 200 µl antibiotic free LB media were added and the reaction tube was incubated at 37 °C with 400 rpm for one hour. Afterwards whole cultures were streaked out onto plates containing Chloramphenicol.

2014.08.31

DNA extraction from GAL4 RFC 25 ONC (1x), PCR 20 (3x), PCR 21 (3x)

template

concentration [ng/µl]

template

concentration [ng/µl]

GAL4 RFC 25 in pSB1C3

154.2

(PCR 21) SEAP I

188.3

(PCR 20) ePDZb I

91.4

(PCR 21) SEAP II

228.3

(PCR 20) ePDZb II

119.4

(PCR 21) SEAP III

209.6

(PCR 20) ePDZb III

113.4

 

 

Test digests

template

enzyme

buffer

fragment size

GAL4 RFC 25 in pSB1C3

EcoRI-HF/PstI-HF

CutSmart

2.0 kb, 0.5 kb

ePDZb

EcoRI-HF/PstI-HF/NotI-HF

CutSmart

mutated:1.0 kb

not mutated: 0.6 kb, 0.4 kb

SEAP

PstI-HF/BbsI

CutSmart

mutated: 4.1 kb, 1.3 kb

not mutated: 4.1 kb, 1 kb, 0.3 kb

Digests pipetted according to protocol.

Description of Image

gel electrophoresis of PCR 21.

Digests of GAL4 and ePDZb were run over 2 % agarose gel. Promega 100 bp was used as DNA ladder. SEAP was run over normal gel, with standard DNA ladder.

Fragment sizes weren’t as expected. Five new colonies for ONC were picked from same plate.

Standardization - September

2014.09.01

No ligation worked. They were repeated with a different protocol.

WPRE RFC 10

0.83 µl

SLC7A1 RFC 10

7.98 µl

P2A RFC 10

0.34 µl

pSB1C3

always 2.75 µl

 

Protocol:             2 µl 10x T4 buffer

                               0.2 µl T4 ligase (4e5 U/ml)

                               add water up to 20 µl

                               incubate at room temperature for 15 minutes

 

Transformed bacteria were streaked out onto plates containing Chloramphenicol and incubated at 37 °C

 

DNA extraction from SEAP ONC (5x)

template

concentration [ng/µl]

SEAP I

215.7

SEAP II

257.1

SEAP III

285.0

SEAP IV

139.9

SEAP V

367.6

Afterwards DNA was digested according to protocol.

template

enzyme

buffer

fragment size

SEAP

PstI-HF/NotI-HF

CutSmart

mutated: 3.7 kb, 1.7 kb

not mutated: 3.7 kb, 1.4 kb, 0.3 kb

 

Description of Image

test digest of overnight culture. Took from agar plate containig PCR 21.

Gel looked fine. SEAP III and SEAP IV will be sequenced. DNA Mini of CAT-1 (2014.08.22.) will be sequenced too, using another sequencing primer.

2014.09.02

Sequencing results of CAT-1 showed that it didn’t contain any PstI site anymore.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

22

ePDZb_mutated

o14sd_010

014sd_020

Phusion

ePDZb_RFC_25_fwd -> AgeI site

0.5 kb

55 °C

23

ePDZb_mutated

o14sd_002

014sd_008

Phusion

ePDZb_AgeI -> ePDZb_RFC_25_rev

0.2 kb

55 °C

24

SEAP III

o14sd_049

014sd_050

Phusion

SEAP_RFC_10

1.6 kb

50 °C

25

SEAP III

o14sd_045

014sd_046

Phusion

SEAP_C2784G_NgoMIV

5.4 kb

50 °C

 

Description of Image

test digest of PCR 22 & 23.

PCR 22 + PCR 23 were done in duplicates, PCR 24 in triplicates. Gel electrophoresis was done in 2 % gel.

All PCR but PCR 23 didn’t show expected size. PCR 23’s DNA fragments were cut out and prepared for gel extraction.

Ligation of CAT-1 and P2A in pSB1C3 didn’t work, but plate with bacteria containing WPRE RFC 10 in pSB1C3 shows eight colonies. They were picked for ONC.

Ligation of CAT-1 and P2a were repeated with a modified yesterday’s protocol. 2 µl of T4 ligase (40,000 U/ml) instead 0.2 µl T4 ligase (400,000 U/ml) were used.

2014.09.03

 

DNA extraction of WPRE ONC (8x)

template

concentration [ng/µl]

template

concentration [ng/µl]

WPRE RFC 10 I

132.9

WPRE RFC 10 V

130.0

WPRE RFC 10 II

148.7

WPRE RFC 10 VI

106.7

WPRE RFC 10 III

91.9

WPRE RFC 10 VII

89.0

WPRE RFC 10 IV

148.8

WPRE RFC 10 VIII

106.7

 

Test digest of WPRE RFC 10

template

enzyme

buffer

fragment size

WPRE

PstI-HF/EcoRI-HF

CutSmart

mutated: 2.0 kb, 0.6 kb

 

Description of Image

test digest of ligated WPRE RFC 10 into pSB1C3.

Digests were run over 1 % agarose gel

All eight test digests looked fine. WPRE RFC V will be sequenced. Other ligations didn’t work perhaps of damaged DNA.

Bacteria were transformed on Ampicillin plate with PCR 25.

2014.09.04

Sequencing results showed that WPRE was correctly ligated into pSB1C3.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

26

CAT-1 mutated

o14sd_031

014sd_032

Q5

CAT-1_C1184A_NgoMIV

5.8 kb

55 °C

27

WPRE RFC 10 V

o14sd_055

014sd_056

Q5

WPRE_C530A_NgoMIV

2.5 kb

55 °C

28

LOV_mutated

o14sd_021

014sd_022

Q5

LOV_RFC_25

3.8 kb

55 °C

29

ePDZb_EcoRI_out

o14sd_021

014sd_022

Q5

ePDZb_RFC_10

3.8 kb

55 °C

30

pKM292

o14sd_011

014sd_012

Q5

GAL4 RFC 25

0.6 kb

50 °C

All PCRs were used to transform bacteria (Ampicillin, but PCR 27 Chloramphenicol) and afterwards incubated at 37 °C.

Triplicates of PCR 30 were made (50 µl) and preparative digest (EcoRI-HF/PstI-HF) was done directly in PCR tube. and afterwards purified. Concentration: 130 ng/µl.

Eight colonies were picked from plate containing PCR 15.

2014.09.05

DNA extraction of ONC PCR 15 (8x) and PCR 25 (5x)

template

concentration [ng/µl]

template

concentration [ng/µl]

P2A I

275.4

SEAP I

248.1

P2A II

192.4

SEAP II

127.1

P2A III

176.3

SEAP III

218.2

P2A IV

339.3

SEAP IV

145.9

P2A V

373.3

SEAP V

167.4

P2A VI

304.2

P2A VII

167.7

P2A VIII

305.7

 

Test digests PCR 15 and PCR 25

template

enzyme

buffer

fragment size

P2A

NgoMIV

CutSmart

mutated: 7.0 kb, 4.7 kb

not mutated: 4.7 kb, 4.1 kb, 2.8 kb

SEAP

NgoMIV

CutSmart

mutated: 5,4 kb

not mutated: 4.4 kb, 1 kb

 

Description of Image

test digest of PCR 15.

P2A and SEAP separated by gel electrophoresis.

Description of Image

test digest of PCR 25.

Test digests of SEAP didn’t work. P2A V + VIII showed fragments with correct size.

Transformations and ligations

No transformation worked.

Following transformations were made:

PCR 26 -29, on Ampicillin plates and incubated at 37 °C

Following ligations were made:

CAT-1 RFC 10, P2A RFC 10, GAL4 RFC 25 in pSB1C3.

2014.09.06

No ligation worked.

2014.09.07

 

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

31

SEAP_PstI_out

o14sd_049

014sd_050

Q5

SEAP RFC 10

1.6 kb

50 °C

32

CAT-1 mutated V

o14sd_031

014sd_032

Q5

CAT-1 RFC 10

1.9 kb

50 °C

33

P2A_mutated_V

o14sd_025

014sd_026

Q5

P2A RFC 25

0.1 kb

55 °C

34

GAL4_binding_domain

o14sd_011

014sd_012

Q5

GAL4 RFC 25

0.6 kb

55 °C

35

WPRE RFC 10

o14sd_055

014sd_056

Q5

WPRE RFC 25

0.6 kb

55 °C

PCR 31-34 were made in 50 µl, PCR 35 in 25 µl.

Description of Image

test digest of PCR 31 & 32.

SEAP looked fine, but CAT-1 showed unspecific PCR products.

Gel extraction of GAL4 binding domain, SEAP, P2A

template

concentration [ng/µl]

GAL 4 binding domain RFC 25

60.8

SEAP RFC 10

27.9

P2A RFC 10

11.9

 

Ligation protocol

plasmid

backbone

P2A

SEAP

GAL 4

length (bp)

2070

136

1577

507

concentration [ng/µl]

27.3

11.9

27.9

60.8

volume [µl]

1.83

0.83

4.10

0.60

water

 

14.83

11.57

15.06

plus 2 µl 1x T4 buffer and 0.5 µl T4 ligase (400,000 U/ml) each

Approaches were incubated at room temperature for 30 minutes.

Transformation: lent cells from our instructor and added 10 µl of ligation approach and 3 µl of PCR 38 respectively.

Transformations were made in duplicates.

2014.09.08

All ligations worked. Three ONC were made from GAL4 RFC 25, P2A RFC 25, SEAP RFC 10, six from PCR 25, eight from WPRE RFC 25.

PCR #

template

primer 1

primer 2

polymerase

product

size

annealing temperature

36

CAT-1_PstI_out

o14sd_039

014sd_040

Q5

CAT-1 RFC 10

1.9 kb

55 °C

37

ePDZb II (145.3 ng/µl)

o14sd_005

014sd_006

Q5

ePDZB_A1775G_EcoRI

3.8 kb

55 °C

PCR according to protocol, afterwards PCRs were digested by DpnI and transformed onto agar plates containing Ampicilin.

2014.09.09

DNA extractions failed due to old buffers. Agar plates were overgrown. Bacteria were diluted onto another plate. Transformation of PCR 36 failed. Repeated it with different primers

PCR #

Template

primer 1

primer 2

polymerase

product

size

annealing temperature

38

CAT-1_PstI_out

o14sd_039

014sd_040

Q5

CAT-1 RFC 10

1.9 kb

50 °C

39

CAT-1_PstI_out

o14sd_039

014sd_040

Q5

CAT-1 RFC 10

1.9 kb

55 °C

 

2014.09.10

Description of Image

test digest of PCR 38 & 39.

Amplifications of CAT-1 RFC 10 were successful. The amount of PCR product at 55 °C was much higher than at 50 °C. Both lanes were cut out used for ligation into pSB1C3.

PCR #

Template

primer 1

primer 2

polymerase

product

size

annealing temperature

40

CAT-1_PstI_out

o14sd_031

014sd_032

Q5

CAT-1_C1184A_NgoMIV

5.8 kb

55 °C

PCR according to protocol, afterwards PCR was digested by DpnI and transformed onto agar plates containing Ampicilin.

Gel extraction of CAT-1

Concentration: 21.4 ng/µl

Ligation approach:

Plasmid

backbone

CAT-1 RFC 10

length (bp)

2070

1925

concentration [ng/µl]

5.1

21.4

volume [µl]

9.8

6.33

Water

11.2

4.37

plus 2 µl 1x T4 buffer and 0.5 µl T4 ligase (400,000 U/ml) each

Control and ligation were incubated at room temperature for 30 minutes. Transformation was done with 10 µl each. Bacteria cultures were incubated at 37 °C and 600 rpm for 60 minutes.

Transformation was done with 100, 50, 20, 10 µl to discover the optimal transformation volume.

Several ONC were made (PCR 31 – 35 (3x each), PCR 37 (10x)).

2014.09.11

DNA extraction from ONC

Template

concentration [ng/µl]

template

concentration [ng/µl]

ePDZB_A1775G_EcoRI I

98.5

ePDZB_A1775G_EcoRI VI

142.1

ePDZB_A1775G_EcoRI II

91.5

ePDZB_A1775G_EcoRI VII

78.1

ePDZB_A1775G_EcoRI III

95.5

ePDZB_A1775G_EcoRI VIII

64.0

ePDZB_A1775G_EcoRI IV

93.8

ePDZB_A1775G_EcoRI IX

80.3

ePDZB_A1775G_EcoRI V

78.0

ePDZB_A1775G_EcoRI X

71.1

GAL4 RFC 25 I

105.9

WPRE_C530A_NgoMIV I

109.3

GAL4 RFC 25 II

123.8

WPRE_C530A_NgoMIV II

119.0

GAL4 RFC 25 III

117.6

WPRE_C530A_NgoMIV III

209.5

P2A RFC 10 I

107.3

SEAP RFC 10 I

117.5

P2A RFC 10 II

117.8

SEAP RFC 10 II

123.4

P2A RFC 10 III

113.1

SEAP RFC 10 III

119.6

 

Test digests

Template

enzyme

buffer

fragment size

P2A RFC 10

EcoRI-HF/PstI-HF

CutSmart

2.0 kb,0.1 kb kb

GAL4 RFC 25

EcoRI-HF/PstI-HF

CutSmart

2.0 kb, 0.5 kb

SEAP RFC 10

EcoRI-HF/PstI-HF

CutSmart

2.0 kb, 1.5 kb

ePDZb

EcoRI-HF/NcoI

CutSmart

mutated: 3.8 kb

not mutated: 3.2 kb, 0.6 kb

 

Gel result

Description of Image

test digest of PCR 31 & 32.

It looked like ePDZb hadn’t been totally digested. The second lane lay short under 1 kb lane and not at 600 bp. WPRE RFC 10, SEAP RFC 10 and P2A didn’t show expected results. Especially it seemed that SEAP RFC 10 was actually identical with GAL 4 RFC 25 due to the lanes at 500 bp, which were expected for GAL4 RFC 25.

As a result GAL4 RFC I will be sequenced

Eight colonies from containing PCR 40 were picked for ONC.

2014.09.12

Despite to bad results on gel electrophoresis, P2A I and SEAP RFC 10 I will be sequenced.

DNA extraction from ONC

Template

concentration [ng/µl]

template

concentration [ng/µl]

CAT-1_C1184A_NgoMIV I

473.8

CAT-1_C1184A_NgoMIV V

269.1

CAT-1_C1184A_NgoMIV II

461.6

CAT-1_C1184A_NgoMIV VI

329.3

CAT-1_C1184A_NgoMIV III

373.3

CAT-1_C1184A_NgoMIV VII

452.2

CAT-1_C1184A_NgoMIV IV

469.1

CAT-1_C1184A_NgoMIV VIII

398.4

 

Test digest

Template

enzyme

buffer

fragment size

CAT-1_C1184A_NgoMIV

XbaI/NgoMIV

CutSmart

mutated: 4.2, 1.2 kb,0.3 kb kb

not mutated: 4.2, 1.2 kb,165 bp, 142 bp.

 

Gel electrophoresis

Description of Image

test digest of PCR 38 & 39.

Samples were run in a 2 % agarose gel. Marker is 100 bp from Promega. Due to missing lane at 300 bp, the restriction site was still in CAT-1.

Ligation of SEAP RFC 10 and CAT-1 were repeated and digested with EcoRI-HF/PstI-HF in CutSmart buffer. Afterwards samples were purified by PCR purification Kit. Concentrations:

Plasmid

backbone

SEAP

CAT-1

length (bp)

2070

1577

1869

concentration [ng/µl]

16.2

39.5

21.4

volume [µl]

1.83

2.79

6.33

Water

 

11.20

7.59

plus 2 µl 1x T4 buffer and 0.5 µl T4 ligase (400,000 U/ml) each

 

         

Transformed and incubated in Chloramphenicol containing LB-medium. 10, 50 and 100 µl of cluture were streaked out onto agar plates.

2014.09.13

Only few colonies were grown. Wait another day before using them for ONC.

Our instructor recommended us to use Pfu-Polymerase instead of Q5 or Phusion for site-directed mutagenisis PCRs.

PCR #

Template

primer 1

primer 2

polymerase

product

size

annealing temperature

40

SEAP_PstI_out (09.01.)

o14sd_045

014sd_046

Pfu

SEAP_G2780C_NgoMIV

5.8 kb

55 °C

41

LOV_09_10

o14sd_039

014sd_040

Pfu

LOV_without_PstI

3.6 kb

55 °C

42

ePDZb (145.3 ng/µl)

o14sd_005

014sd_006

Pfu

ePDZb_Eco_out

3.4 kb

55 °C

43

CAT-1 mutated

o14sd_037

014sd_038

Pfu

CAT-1_AgeI_out

5.8 kb

55 °C

44

WPRE RFC 10

o14sd_059

014sd_060

Pfu

WPRE_NgoMIV_out

2.6 kb

55 °C

45

P2A RFC 10 (107.3 ng/µl)

o14sd_027

014sd_028

Pfu

P2A_NgoMIV_out

2.2 kb

55 °C

 

Pfu protocol:     basically just a normal PCR program:      1x           95 °C      30 seconds

                                                                                                              15x:       95 °C      30 seconds

                                                                                                                             55 °C      1 minute

                                                                                                                             68 °C      2 minutes/kb

                                                                                                              1x:          68 °C      10 minutes

Afterwards, PCRs weren’t digested by DpnI, but 5 µl were taken for transformation on either Ampicillin or Chloramphenicol agar plates.

2014.09.14

3 transformations worked (PCR 40, 42, 44). Colonies of PCR 42 were small, so they were left an additional day in the incubator.

2014.09.15

PCR #

Template

primer 1

primer 2

polymerase

product

size

annealing temperature

46

GAL4

o14sd_011

014sd_012

Q5

GAL4 RFC 25

0.5 kb

55 °C

PCR was made in 50 µl and in triplicates.

2014.09.16

Triplicates of PCR 46 were digested in CutSmart by EcoRI-HF/PstI-HF for ligation into pSB1C3. Ligation of PCR 46 was made as last time reported.

ONC were made from PCR 40 (2x), 42 (6x), 44 (2x).

2014.09.17

DNA extraction of ONCs.

Template

concentration [ng/µl]

Template

concentration [ng/µl]

ePDZb_Eco_out I

135.6

ePDZb_Eco_out VI

313.9

ePDZb_Eco_out II

185.2

SEAP_NgoMIV_out I

151.0

ePDZb_Eco_out III

62.0

SEAP_NgoMIV_out II

200.0

ePDZb_Eco_out IV

117.0

WPRE_NgoMIV_out I

84.0

ePDZb_Eco_out V

63.9

WPRE_NgoMIV_out II

115.8

 

Test digests

Template

enzyme

buffer

fragment size

WPRE_NgoMIV_out

XhoI/NgoMIV

CutSmart

????

SEAP_NgoMIV_out

NgoMIV_BamHI-HF

CutSmart

Mutated: 3 kb, 1.7 kb

Not mutated: 3 kb, 0,9 kb, 0.8 kb

ePDZb

EcoRI-HF/XbaI/NheI

CutSmart

mutated: 2.4 kb, 1.4 kb

not mutated: 2.4 kb, 0.9 kb. 0.5 kb

 

Ligation of GAL 4 RFC 25 didn’t work.

2014.09.18

Every test digest failed.

Ligation of GAL4 RFC 25 repeated, but with Quick ligation buffer.

Plasmid

backbone

GAL4 RFC 25

length (bp)

2070

441

concentration [ng/µl]

64.1

87.9

volume [µl]

0.78

0.36

Water

 

1.20

plus 2.6 µl 1x T4 buffer and 0.2 µl T4 ligase (400,000 U/ml)

We used for a 2nd approach instead of molare ratio of 1:3 just thrice volume of insert (0.73 µl pSB1C3 + 1.47 µl GAL 4 RFC 25).

Ligations were incubated at room temperature for 15 minutes. 5 µl were taken for transformation and afterwards plates were incubated at 37 °C.

PCR #

Template

primer 1

primer 2

polymerase

product

size

annealing temperature

47

pKM292 re-transformation

o14sd_011

014sd_012

Q5

GAL 4 RFC 25

0.6 kb

55 °C

48

p14zl_003

o14sd_029

014sd_030

Q5

Puromycin acetyl transferase

3.6 kb

55 °C

49

WPRE RFC 10 seq.

o14sd_059

014sd_060

Q5

WPRE RFC 10 with RFC 25 overhangs

0.6 kb

55 °C

Each PCR was made in 50 µl and triplicates.

Gel electrophoresis showed fine results. Lanes were cut out and ligations into pSB1C3 were made at 21.09.

2014.09.21

 

PCR #

Template

primer 1

primer 2

polymerase

product

size

annealing temperature

50

CAT-1 mutated

o14sd_041

014sd_042

Pfu

CAT-1 RFC 10 with RFC 25 overhangs

1.9 kb

55 °C

51

SEAP_PstI_out

o14sd_051

014sd_052

Pfu

SEAP RFC 10 with RFC 25 overhangs

1.6 kb

50 °C

Each PCR was made in 50 µl and triplicates.

ONC were made from ligations.

 

2014.09.22

 

No PCR product visible. PCR 50 & 51 will be repeated with Phusion instead of Pfu with a common annealing temperature of 50 °C.

PCR #

Template

primer 1

primer 2

polymerase

Product

size

annealing temperature

54

PCR 1.1 II

o14sd_005

014sd_006

Phusion

ePDZb_EcoRI_out

1.9 kb

55 °C

PCR was digested by DpnI and afterwards 3 µl were taken for transformation. The bacteria culture was streaked out onto a Ampicilin plate.

 

DNA extraction of ONC

Template

concentration [ng/µl]

Template

concentration [ng/µl]

WPRE RFC 10 (PCR 49) I

105.0

Puromycin acetyltransferase RFC 25 I

131.4

WPRE RFC 10 (PCR 49) II

146.0

Puromycin acetyltransferase RFC 25 II

161.2

WPRE RFC 10 (PCR 49) III

131.0

SEAP_NgoMIV_out II Puromycin acetyltransferase RFC 25 III

70.0

WPRE RFC 10 (PCR 49) IV

267.0

Puromycin acetyltransferase RFC 25 IV

124.0

WPRE RFC 10 (PCR 49) V

140.0

Puromycin acetyltransferase RFC 25 V

230.0

GAL 4 RFC 25 I

156.1

GAL 4 RFC 25 II

247.8

GAL 4 RFC 25 III

134.4

GAL 4 RFC 25 IV

135.7

GAL 4 RFC 25 V

130.4

Test digsts of ONC

Template

enzyme

buffer

fragment size

WPRE RFC 25

NcoI-HF

CutSmart

1.5 kb, 1.2 kb

GAL 4 RFC 25

XhoI

CutSmart

1.2 kb, 0.9 kb, 0.3 kb

Puromycin acetyltransferase

XhoI

CutSmart

1.8 kb, 0.9 kb

 

All digests showed lanes with the expected sizes.

2014.09.23

 

Transformation with PCR 54 worked. Three colonies were picked for ONC.

Gel electrophoresis of PCR 50 & PCR 51

Description of Image

test digest of PCR 50 & 51.

Gel electrophoresis of PCR 50 & PCR 51

img src="http://2014.igem.org/wiki/images/2/2a/Freiburg2014_2014-09-23_testverdauPCR50_PCR51_neu.jpg" alt="Description of Image">

test digest of PCR 50 & 51.

2014.09.24

 

DNA extraction of ONC

Template

concentration [ng/µl]

ePDZb_Eco_out I

49.6

ePDZb_Eco_out II

67.7

ePDZb_Eco_out III

52.7

 

Test digest

Template

enzyme

buffer

fragment size

ePDZb_EcoRI_out

EcoRI-HF/ClaI/HindHIII-HF

CutSmart

Mutated: 2.3 kb, 1.5 kb

Not mutated: 2.3 kb, 0.9 kb, 0.6 kb

 

Result: all 3 samples showed expected DNA lanes. Colony 2 will be sequenced.

Colony 1 of WPRE RFC 25, GAL 4 RFC 25 and Puromycin acetyltransferase RFC 25 will be sequenced.

 

2014.09.25

 

All biobricks showed expected DNA sequence and ePDZb lost the EcoRI site.

PCR #

Template

primer 1

primer 2

Polymerase

Product

size

annealing temperature

55

ePDZb_EcoRI_out

o14sd_001

014sd_002

Phusion

ePDZb_AgeI_out

3.8 kb

55 °C

56

SEAP_PstI_out (367.6 ng/µl)

o14sd_045

014sd_046

Phusion

SEAP_NgoMIV_end

5.8 kb

50 °C

57

SEAP_PstI_out (367.6 ng/µl)

o14sd_049

014sd_050

Phusion

SEAP RFC 10

1.6 kb

50 °C

58

WPRE RFC 10 (25.09.)

o14sd_055

014sd_056

Phusion

WPRE RFC 25

1.6 kb

50.5 °C

 

Gel of PCR 57 showed a strong lane. The lane was cut out and prepared for digest/ligation. Digest was done with EcoRI-HF/Pst-HF in CutSmart.

Ligation of PCR 50 and PCR 57

 

Plasmid

backbone

CAT-1 RFC 25 overhangs

SEAP RFC 10

length (bp)

2070

1945

1577

concentration [ng/µl]

64.1

68.7

80.4

volume [µl]

0.38

1.84

1.71

Water

 

1.20

 

plus 2.6 µl 1x T4 buffer and 0.2 µl T4 ligase (400,000 U/ml)

Additional approach: just use a triple volume of insert each (0.6 µl backbone, 1.8 µl insert).

2014.09.26

Transformation of PCR 58 didn’t work but PCR 55 & 56 showed colonies on plates. 5 colonies were picked for ONC.

Ligations were partly successful. There for both SEAP RFC 10 and CAT-1 with RFC 25 overhangs colonies. 5 of them were picked for ONC each.

2014.09.27

 

DNA extraction from OVC

Template

concentration [ng/µl]

Template

concentration [ng/µl]

PCR 56 I

160.4

PCR 55 I

81.1

PCR 56 II

122.9

PCR 55 II

71.4

PCR 56 III

109.9

PCR 55 III

46.8

PCR 56 IV

105.2

PCR 55 IV

43.1

PCR 56 V

199.3

PCR 55 V

81.0

SEAP RFC 10 I

182.2

PCR 50 I

130.2

SEAP RFC 10 II

100.9

PCR 50 II

120.2

SEAP RFC 10 III

122.7

PCR 50 III

17.2

SEAP RFC 10 IV

239.0

PCR 50 IV

128.2

SEAP RF 10 V

201.6

PCR 50 V

170.3

 

Test digests

Template

enzyme

buffer

fragment size

PCR 50

EcoRV-HF/KpnI-HF

CutSmart

2.2 kb, 1.3 kb

PCR 55

AgeI-HF/XbaI/NheI

CutSmart

mutated: 2.4 kb, 1.4 kb

not mutated: 2.4 kb, 0.9 kb, 0.5 kb

PCR 56

NgoMIV/BamHI-HF

CutSmart

mutated: 3.0 kb, 1.7 kb

not mutated: 3.0 kb, 1 kb, 0.7 kb

PCR 57

NcoI-HF

CutSmart

1.8 kb, 1 kb, 0.7 kb

 

Gel electrophoresis

Description of Image

test digest of PCR 50, 55-57.

PCR 50, 56 and 57 failed. PCR 55 looked like the plasmid had been cut once. Sequence analysis showed that XbaI site was overlapped with a methylation site, thus restriction site was blocked and couldn’t recognized anymore. Therefore PCR 55 I would be sequenced.

2014.09.28

PCR #

Template

primer 1

primer 2

Polymerase

Product

size

annealing temperature

59

WPRE RFC 10 -NgoMIV

o14sd_055

014sd_056

Phusion

WPRE RFC 25

0.6 kb

57 °C

60

CAT-1 PstI out

o14sd_039

014sd_040

Phusion

CAT-1 RFC 10

1.9 kb

56 °C

PCR 59 was done in 50 µl approaches and in triplicates and was separated in a 2 % gel.

Gel electrophoresis

 

Description of Image

test digest of WPRE RFC 25 ligation into pSB1C3.

Results looked fine. The PCR product was cut out with EcoRI-HF/PstI-HF and used for ligation into pSB1C3.

Description of Image

test digest of PCR 60.

Results looked fine. The PCR product was cut out with EcoRI-HF/PstI-HF and used for ligation into pSB1C3.

 

Gel extraction

Template

concentration [ng/µl]

WPRE RFC 25

76.2

CAT-1 RFC 10

289.6

SEAP RFC 10

like PCR 57

 

2014.09.30

Sequencing results confirmed that ePDZb is AgeI-free.

PCR #

Template

primer 1

primer 2

Polymerase

Product

size

annealing temperature

61

PCR 55 I

o14sd_007

014sd_008

Phusion

ePDZb RFC 10

0.6 kb

56.5°C

62

SEAP PstI out

o14sd_045

014sd_046

Phusion

SEAP_C2784G_NgoMIV

5.4 kb

56 °C

PCR Product was purified by gel extraction and cut with EcoRI-HF/PstI-HF and used for ligation into pSB1C3.

PCR 62 was digested by DpnI and transformed on agar plate containing Ampicillin.

ligation

 

WPRE RFC 25 (09/29)

CAT-1 RFC 10 (09/29)

SEAP RFC 10 (09/25)

pSB1C3

1.46 µl

1 µl

1 µl

insert

0.74 µl

0.4 µl

0.4 µl

water

/

0.8

/

plus 2.6 µl Quick ligation buffer, 0.2 µl T4 ligase (400,000 U/ml)

 

2014.10.01

All transformed plates had colonies.

DNA extraction

Template

concentration [ng/µl]

WPRE RFC 25 I

130.0

WPRE RFC 25 II

109.0

WPRE RFC 25 III

115.0

WPRE RFC 25 IV

140.0

PCR 62 I

448.0

PCR 62 II

458.0

PCR 62 III

490.0

PCR 62 IV

413.0

 

Template

concentration [ng/µl]

Template

concentration [ng/µl]

SEAP RFC 10 I

216.9

CAT-1 RFC 10 I

265.9

SEAP RFC 10 II

192.9

CAT-1 RFC 10 II

396.3

SEAP RFC 10 III

193.3

CAT-1 RFC 10 III

223.2

SEAP RFC 10 IV

176.5

CAT-1 RFC 10 IV

272.2

SEAP RFC 10 V

167.5

CAT-1 RFC 10 V

209.0

SEAP RFC 10 VI

193.4

CAT-1 RFC 10 VI

188.6

ePDZb RFC 10 I

125.1

ePDZb RFC 10 II

144.4

ePDZb RFC 10 III

117.5

ePDZb RFC 10 IV

125.0

ePDZb RFC 10 V

118.8

ePDZb RFC 10 VI

109.5

Test digests

Template

enzyme

buffer

fragment size

PCR 59

EcoRV-HF/NgoMIV

CutSmart

1.7 kb, 0.9 kb

PCR 60

EcoRV-HF/KpnI-HF

CutSmart

2.2 kb, 1.4 kb, 0.4 kb

PCR 61

XhoI

CutSmart

1.8 kb, 0.9 kb

PCR 62

BamHI-HF/NgoMIV

CutSmart

2.3 kb, 1 kb, 0.3 kb

Gel electrophoresis

Description of Image

test digest of PCR 62 and ligation of WPRE RFC 25 into pSB1C3.

All results looked fine. It seemed that SEAP contained a NgoMIV site less and the WPRE digests proved that WPRE RFC 25 is in pSB1C3.

Description of Image

test digest of following ligations: ePDZb RFC 10, SEAP RFC 10, CAT-1 RFC 10.

Results looked fine. Every sample showed expected lane size.

Colony one of each plasmid was sent to sequencing.

Standardization - October

2014.10.02

All four ligations contain expected DNA sequence.