Standardization

Standardization - August

PCR mix for site-directed mutagenisis (25 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

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

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.

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.

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:

test digest with:

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:

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.

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)

Test digest with:

test digest was done overnight.

2014.08.25

Result gel electrophoresis (digest CAT-1)

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)

Test digests according to 2014.08.23 overnight.

2014.08.27

Gel electrophoresis from test digest 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 15 was done in 50 µl approach and triplicates.

Gel electrophoresis from 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.

Gel electrophoresis from 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

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

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)

Test digests PCR 1.1 I & PCR 3.1 III:

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 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

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.

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.)

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

ratio backbone:insert = 1:3

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)

Test digests

Digests pipetted according to protocol.

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.

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)

Afterwards DNA was digested according to protocol.

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 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)

Test digest of WPRE RFC 10

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.

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)

Test digests PCR 15 and PCR 25

P2A and SEAP separated by gel electrophoresis.

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

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

Gel extraction of GAL4 binding domain, SEAP, P2A

Ligation protocol

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 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

2014.09.10

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 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:

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

Test digests

Gel result

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

Test digest

Gel electrophoresis

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:

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.

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 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.

Test digests

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.

We used for a 2^nd 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.

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

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 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

Test digsts of ONC

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

Gel electrophoresis of PCR 50 & PCR 51

2014.09.24

DNA extraction of ONC

Test digest

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.

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

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

Test digests

Gel electrophoresis

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

Gel electrophoresis

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

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

Gel extraction

2014.09.30

Sequencing results confirmed that ePDZb is AgeI-free.

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

2014.10.01

All transformed plates had colonies.

DNA extraction

Test digests

Gel electrophoresis

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.

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.