29-04 - 05-05-13

Generation of Plasmid Backbones

Transformation of Biobricks

• Added 10 µl H₂O to each well
• Incubated for 10 min at RT
• Thawed 5x chemical competent E.coli Top10
• 3 µl of plasmid DNA were added
• Incubated for 10 min on ice
• Heat shock for 40 s at 42.2°C
• Incubated for 10 min on ice
• Added 500 µl LB Medium
• Incubated at 37°C for 40 min
• Centrifuged 120 at 5,000 rpm, supernatant discarded
• Pellet resuspended in remaining medium
• Plated on plates with the corresponding antibiotics (as shown in the table above, section: resistances)
• Incubated for 2 days at RT
• One colony was picked from each plate and incubated over night at 37°C in LB medium with the antibiotic listed above

Result

All 5 parts from the Registry Distribution 2012 were successfully transformed in E.coli Top10 except for the one containing the AraC promotor.

Amplification of DelH Fragments

Design of Primers

13-05 - 19-05-13

Amplification of DelH F1

Gel Purification

45 µl of DelH F1 PCR (08-05) product were run on a 0,8% agarose gel (1 h with 135V). Gel extraction was performed using gel extraction kit of Qiagen, and diluted in 25 µl ddH₂O.

Result

=> Because the concentration is so low, a re-PCR of the PCR product (08-05) is going to be done.

re-PCR Conditions F1.W3.A

Result

Expected length: 10 Kb

Fig.3.1 PCR of DelH fragment 1 (loaded 20 µL)
l1:fragment 1 of DelH, l2: 2log ladder

Different unspecific bands were observed.

=> Test digest to confirm identity.

Test Restriction Digest

Afterwards, purification with the nucleotide removal kit was performed, but due to using wrong column, there was no product left.

=> Repeat amplification of DelH F1.

PCR Conditions F1.W3.B

Result

Expected length: 10 Kb

Fig.3.2 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2-4: fragment 1 of DelH, l5: fragment 2 of DelH

There is only an unspecific band at the second PCR of F1 with 2.5 µl DMSO.

=> Repeat using DMSO and altered PCR program.

PCR Conditions F1.W3.C

Result

Expected length: 10 Kb

Fig.3.3 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 1 of DelH, l3: fragment 2 of DelH, l4: fragment 1 of DelH, l5: fragment 2 of DelH

There was no band visible.

=> Repeat using DMSO and altered PCR program.

Amplification of DelH F2

Gel Purification

45 µl of DelH F1 PCR (07-05) product were run on a 0,8% agarose gel (1 h with 135V). Gel extraction was performed using gel extraction kit of Qiagen, and diluted in 25 µl ddH₂O.

Result

=> Because the concentration is so low, a re-PCR of the PCR product (07-05) is going to be done.

Re-PCR Conditions F2.W3.A

Result

Expected length: 8 Kb

Fig.3.4 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 2 of DelH

Specific band was observed.

=> Test digest to confirm identity.

Test Restriction Digest

Afterwards, a purification with the nucleotide removal kit was performed, but due to using wrong column, there was no product left.

=> Repeat amplification of DelH F2.

PCR Conditions F2.W3.B

Result

Expected length: 8 Kb

Fig.3.2 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 1 of DelH, l3: fragment 2 of DelH

There is no band visible.

=> Repeat using altered PCR program.

PCR Conditions F2.W3.B

Result

Expected length: 8 Kb

Fig.3.3 PCR of DelH fragment 1 (loaded 20 µL)
l1:2log ladder, l2: fragment 1 of DelH, l3: fragment 2 of DelH

There was a specific band at 8 Kb

=> Fragment was cut and gel extracted.

Generation of Backbone pSB6A1-AraC-lacZ

Miniprep of Amplified Parts

DH10ß containing I13453 and K20600 (for AraC) and backbone pSB1AK3 (lacZ) were grown ON at 37°C and minipreped.

Result

Restriction Digest

• AraC was cut with EcoRI-HF and SpeI buffered in NEB4
• LacZ was cut with XbaI and PstI buffered in NEB3

• Incubated for 1h at 37°C
• Purified with Qiagen Kit and diluted in 20 µl ddH₂O

Result

Ligation of pSB6A1, AraC and lacZ

• Incubated at RT for 45 min
• Chemical transformation of competent DH10ß
• Streaked on LB Amp plates
• Incubation ON at 37°C

Miniprep of pSB6A1-AraC-lacZ

• One colony each was picked and grown in 2 ml LB Amp ON
• Cultures were minipreped.

Result

Restriction Digest of Backbone pSB6A1-AraC-lacZ

• AraC-lacZ was cut from pSB6A1-AraC-lacZ using PstI & EcoRI-HF

• Incubated for 1 h at 37°C
• Purified with Qiagen kit and diluted in 20 µl ddH₂O

Result

Ligation of AraC-lacZ with pSB1C3

• Incubated at RT for 50 min
• Heat inactivation at 70°C for 5 min
• 10 µl were used for a chemical transformation in TOP10 and plated on LB Chlor
• Incubation ON at 37°C

Conlony-PCR Conditions BB.W3.A

Result

Expected length:

Fig.3.5 PCR of BB psB1C3-AraC-lacZ(loaded 20 µL)
l1:2log ladder, l2: psB1C3-AraC-lacZ
no yield

There was no fragments visible.

=> Is picked coloniy negative or did entire ligation not work out?

20-05 - 26-05-13

DelH Fragment F1a

• The new primer arrived.

PCR Conditions F1a.W4.A

Results

Expected band: 5 Kb

Fig.4.1 PCR ofDelH-fragments and pSB1C3(loaded 20 µL)
l1:2log ladder, l2-3: DelH-F1a without DMSO, l4-5: DelH-F1a with DMSO, l6-7: DelH-F1b, l8:2log ladder, l9:pSB1C3 l10: pSB1C3-AraC-lacZ

The gel shows a lot unspecific bands for both DelH F1a samples.

=> Alter PCR conditions.

DelH F1b

• The new primer arrived

PCR Conditions F1b.W4.A

Result

Expected band: 5 Kb

Fig.4.1 PCR ofDelH-fragments and pSB1C3(loaded 20 µL)
l1:2log ladder, l2-3: DelH-F1a without DMSO, l4-5: DelH-F1a with DMSO, l6-7: DelH-F1b, l8:2log ladder, l9:pSB1C3 l10: pSB1C3-AraC-lacZ

The gel shows a lot unspecific bands for DelH F1b, but also the band at 5 Kb.

=> Band was cut and gel isolated.

Generation of Backbone pSB6A1-AraC-lacZ

Miniprep

• From three different clones, 2ml of pSB6A1-AraC-lacZ (6) in LB Amp were minipreped and eluted in 30 µl ddH₂O. Additionally, a glycerol stock was prepared.

Result

PCR Conditions BB.W4.A

Results

Expected band: 7.4 Kb

Fig.4.2 PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL)
l1:2log ladder, l2-3: pSB6A1-AraC-lacZ (8),l4:2log ladder, l5-6: pSB6A1-AraC-lacZ (10)
no band visible

The gel shows no bands.

=> Perform test restriction to test identity of plasmid.

Restriction Digest A

• Digest with PstI & XbaI of samples 8, 9 and 10 for checking identity of the construct

• Incubated for 1 h at 37°C

Result

Expected bands: 3360 and 4022 bp

Fig.4.3 PCR of BB pSB6A1-AraC-lacZ (loaded 50 µL)
l1:2log ladder, l2: pSB6A1-AraC-lacZ (8), l3: pSB6A1-AraC-lacZ (9), l4: pSB6A1-AraC-lacZ (10),l5:2log ladder, l6: pSB6A1-AraC-lacZ (8) digested with XbaI & PstI, l7: pSB6A1-AraC-lacZ (9) digested with XbaI & PstI, l8: pSB6A1-AraC-lacZ (10)digested with XbaI & PstI, l9:2log ladder, l10: pSB6A1-AraC-lacZ (8) 1:10 diluted, l11: pSB6A1-AraC-lacZ (10) 1:10 diluted, l12: pSB1C3

The gel shows a band at 5 Kb.

=> Colonies 8, 9 and 10 are not the desired construct.

Restriction Digest B

• Digest with KpnI of samples 8, 9, 10 for checking length of the construct and KpnI and BamHI to check for identity.

Result

• Expected bands: 7.381 bp and 4.607 bp & 2.781 bp

Fig.4.4 BB pSB6A1-AraC-lacZ test digested (loaded 50 µL)
l1:2log ladder, l2: pSB13-AraC-lacZ (8), l3: pSB6A1, l4-6: pSB6A1-AraC-lacZ digested with BpsI, l7:2log ladder, l8: pSB13-AraC-lacZ (8), l9: pSB6A1, l10-12: pSB6A1-AraC-lacZ digested with BpsI & BamHI

=> Colonies 8, 9 and 10 are not desired construct. Pick new colony from pSB1C3-AraC(6)-lacZ and test digest.

Miniprep

A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed.

Result

pSB6A1-AraC(6)-lacZ construct : 94 [ng/µl]

Test Restriction Digest

The new miniprep as well as one of the wrong pSB6A1-AraC(6)-lacZ ones were restriction digested with EcoRI-HF and PstI.

• Incubated 1 h at 37°C

Result

Expected bands:  ? Kb

Fig.4.5 Test digest of pSB1C3-AraC-lacZ with EcoRI & PstI (loaded 20 µL)
l1:2log ladder, l2: pSB1C3-AraC-lacZ

The gel showed again these three bands (2.070 and 3.360 and ~4 Kb)

=> Colonies 8, 9 and 10 are not the desired construct.

Generation of Backbone pSB1C3-AraC-lacZ

Test Restriction Digest

In order find out why pSB6A1-AraC-lacZ shows wrong restriction pattern, parental pSB1C3-AraC-lacZ was restriction digested using KpnI as well as KpnI & BamHI.

Result

Expected bands: 5.436 bp and 4.906 bp & 530 bp

Fig.4.4 BB pSB6A1-AraC-lacZ test digested (loaded 50 µL)
l1:2log ladder, l2: pSB13-AraC-lacZ (8), l3: pSB6A1, l4-6: pSB6A1-AraC-lacZ digested with BpsI, l7:2log ladder, l8: pSB13-AraC-lacZ (8), l9: pSB6A1, l10-12: pSB6A1-AraC-lacZ digested with BpsI & BamHI

=> pSB1C3-AraC(6)-lacZ is not desired construct. Pick new colony and test digest.

Miniprep

A new colony was picked from pSB1C3-AraC(6)-lacZ plate and a mini prep was performed.

Result

pSB1C3-AraC(6)-lacZ construct : 203 [ng/µl]

Test Restriction Digest

The new miniprep as well as one of the wrong pSB6A1-AraC(6)-lacZ ones were restriction digested with EcoRI-HF and PstI.

• Incubated 1 h at 37°C

Result

Expected band:  ? Kb

Fig.4.6 BB digested with EcoRI & PstI (loaded 20 µL)
l1:2log ladder, l2: digested pSB1C3-AraC-lacZ, l3: digested pSB6A1-AraC-lacZ

Gel showed again the three bands (2070 and 3360 and ~4 Kb).

=> pSB1C3-AraC(6)-lacZ is not desired construct. Start assembly all over again.

Test Restriction Digest pSB6A1-J04450

pSB6A1-J04450 was test digested to check for identity.

Result

Fig.4.4 BB pSB6A1-AraC-lacZ test digested (loaded 50 µL)
l1:2log ladder, l2: pSB13-AraC-lacZ (8), l3: pSB6A1, l4-6: pSB6A1-AraC-lacZ digested with BpsI, l7:2log ladder, l8: pSB13-AraC-lacZ (8), l9: pSB6A1, l10-12: pSB6A1-AraC-lacZ digested with BpsI & BamHI

This means:

• The chloramphenicol backbone has the right length.
• The digested chloramphenicol backbone is also ok.
• The digested fragment pSB6A1-AraC-lacZ (with PstI & EcoRI-HF) showed the 2 expected bands at 2,070 bp and 3,360 bp, but another band around ~4 Kb.

=> Something is not ok with this ligated construct and we have to start all over again.

27-05 - 02-06-13

Generation of pSB6A1

Miniprep

• Grow pSB6A1 from glycerol stock in 2 ml LB Amp ON at 37°C.
• Miniprep using miniprep kit from Qiagen and dilute in 30 µl ddH₂O.

Result

Concentration 69.5 ng/µl

Digest

• The pSB6A1 was generated from the parts registry and cut with EcoRI & PstI

The digest was incubated 45 min at 37°C.

Result

Expected band: 3985 bp (pSB6A1) and at 1106 bp (insert=J04450)

Fig.5.1 Generation of pSB6A1 for Backbone generation (loaded 50 µL of PCR)
l1:2-log ladder, l2-3:digested pSB6A1
expected band at 4 Kb was cut out

=> Expected bands are present. Backbone pSB6A1 was cut out and gel purified (c=4 ng/µl).

Generation of AraC Fragment

Gel Extraction

Gel extraction was performed with the gel extraction kit from Qiagen of gel slice (cut 24-05) and was eluted in 30 µl.

Result

The measured concentrations are shown in the table below and was stored at -20°C.

Generation of lacZ Fragment

Gel Extraction

Gel extraction was performed with the gel extraction kit from Qiagen of gel slice (cut 24-05) and was eluted in 30 µl.

Result

The measured concentrations are shown in the table below and was stored at -20°C.

Generation of Backbone pSB6A1-AraC-lacZ

Ligation of pSB6A1 with AraC-lacZ

A ligation was performed of fragments for 1 h at RT.

• The ligase was inactivated by heat shock for 5 min at 70°C.

Transformation

• Afterwards, a transformation was performed with 15 µl of the ligation reaction in competent TOP10.
• Cells were streaked on LB Amp plates and incubated ON at 37°C.

Induction

• The following day, 5 white colonies were picked (because the red ones still have the mRFP insert J04450) and were incubated in 2 ml LB Amp with 20 µl Arabinose (10%) and 100 µl X-Gal (20 ng/µl).

Result

2 of the colonies turned blue

=> Perform miniprep.

Miniprep

Miniprep was performed according to Qiagen's instructions.

Result

DelH F1a

PCR Conditions F1a.W5.A

Result

Expected band: 5 Kb

Fig.5.2 PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR)
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2

The gel shows a lot unspecific bands for both DelH F1a samples.

=> Fragment was cut and gel purified.

DelH F1b

PCR Conditions F1b.W5.A

Result

Expected band: 5 Kb

Fig.5.2 PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR)
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2

Gel shows one band at the expected length of 5 Kb, but also other side bands below and a huge, bright band at 2 Kb.

=> Band was cut and gel isolated.

DelH F2

PCR Conditions F2.W5.A

Result

Expected length: 8 Kb

Fig.5.2 PCR of DelH fragment F1a, F1b & F2(loaded 50 µL of PCR)
l1:2-log ladder, l3-4:F1a,l5-6:F1b,l7-8:F2

There was a specific band at 8 Kb.

=> Fragment was cut and gel extracted.

03-06 - 09-06-13

Generation of DelH plasmid pHM01

Elongation-PCR Conditions BB.W6.A

The restriction sites were added to pSB6A1-AraC-lacZ via PCR.

Result

Expected band: 7.4 Kb

Fig.6.1 PCR of pSB6A1-AraC-lacZ & digest of pSB6A1-AraC-lacZ (loaded 5 & 20 µL of PCR)
l1: 2log ladder, l2-3: PCR of BB, l4-5: digested BB of EcoRI & PstI
PCR and digest sho the expected band at 7.4 Kb but also unspecific side bands

Gel shows expected band.

=> Band was cut and gel extracted.

Test Restriction Digest

Isolated fragment was test digested to confirm identity.

• The test digest was incubated 1 h at 37°C.

Result

Expected band: ~4 Kb & 3.4 Kb

Fig.6.1 Digest of pSB6A1-AraC-lacZ (loaded 5 & 20 µL of PCR)
l1: 2log ladder, l2-3: PCR of BB, l4-5: digested BB of EcoRI & PstI
PCR and digest sho the expected band at 7.4 Kb but also unspecific side bands

Gel shows expected band.

=> Backbone is fine.

Restriction Digest of pSB6A1-AraC-lacZ for Ligation

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=24 ng/µl

Restriction Digest of DelH F1a for Ligation

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=9 ng/µl

Restriction Digest of DelH F1b for Ligation

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=19 ng/µl

Restriction Digest of DelH F2 for Ligation

Restriction Digest was purified with Qiagen nucleotide removal kit.

Result

c=9 ng/µl

Ligation of pHM01:DelH-pSB6A1-AraC-lacZ

Ligations was performed of all 4 fragments (digested and purified) for 1 h at RT in 2 different mixes.

• The ligase was inactivated by heat shock for 5 min at 70°C.
• The reaction mix was purified with Qiagen nucleotide removal.

Result

Electroporation

Two electroporations were performed.

• 20 ng
• 30 ng

• After an incubation time of 1 h in SOC-medium, cells were centrifuged and 10 µl plated on one LB Amp plate (with Arabinos and X-Gal) and 100 µl on another plate prepared with the same conditions.
• Plates were incubated ON at 37°C.

Result

White colonies grew on the plates.

Colony PCR

6 colonies of plate E1 (colonies 1-6) and 6 colonies of plate E2 (7-12) as well as ligation mixes 1 & 2 were checked by PCR.

Result

Expected length: 663 bp.

Fig.6.2 Colony PCR of DelH-construct (loaded 5 µL)
l1:2log ladder, l2-10:Colony PCR of colonies 1-9, l10:2 log ladder, l11-15:Colony PCR of colonies 1-9
l2-3: shows expected band at 663 bp

None of the colonies shows a band. Ligation mixes 1 and 2 show the expected band at ~600 bp.

=> The ligation worked, but none of the correct plasmids were present in the analyzed colinies.

10-06 - 16-06-13

Characterization of DelH plasmid of 02-06

Re-PCR of DelH fragment F1a & F1b

Result

Expected band: 5 Kb.

Fig.7.1 Gel of Re-PCR of DelH-F1a and DelH-F1b (loaded 20 µL)
l1 2 log ladder, l2F1a, l3 F1b
no specific band at expected 5 Kb

There are no specific bands at 5 Kb.

=> We therefore failed to reamplify DelH F1a and F1b from the PCR product.

=> Additionally, in the elctroporated colonies, there is no fragment F1a and F1b.

=> We need to design new forward primers for DelH F1a & F1b.

Amplification of DelH F1a

New Primers

PCR Conditions F1a.W7.A

Because the annealing temperature of DelH_f1_fw_long is 77,4°C and of the primer DelH_EcoRI_rev is 69,6°C, a 2-step PCR was performed.

• Using hot start at 98°C

Result

Expected band: 5 Kb

Fig.7.2 Gel of amplified DelH-1a fragment with different fw primern (loaded 20 µL)
l1 2 log ladder, l2F1a, l3 F1a
l2 DelH-1a fragment shows a specific band ~ 5 Kb

There is a specific band at 5 Kb for DN11. The other primers show only unspecific products.

=>The DN11 primer will be used. Using PCR conditions F1a.W7.A and short2 primer, the PCR was repeated to produce more product.

17-06 - 23-06-13

Amplification of DelH F1a

Elongation-PCR Conditions F1a.W8.A

• Using hot start at 98°C

Result

Expected band: 5 Kb

Fig.8.1 gel of amplified fragments (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1a
l2: DelH-F1a shows specific band = cut out

Gels shows expected band.

=> Band was cut and gel extracted.

Restriction Digest

DelH F1a was restricted with EcoRI & PacI.
Afterwards it was purified with nucleotide removal kit and DNA concentration was measured.

Result

Expected band: 5 Kb

Fig.8.2 Gel of digested F1a with (loaded 20 µL)
l1: 2 log ladder, l2-3: DelH-F1a digested

Gel shows expected band at ~5 Kb.

=> Restriction digest mix was purified by precipitation.

Purification of Restriction Digest

• Add 1 ml isopropanol
• Centrifuge 20 min full speed
• Discard supernatant
• Add 750 µl 70% ethanol
• Centrifuge 5 min full speed
• Discard supernatant
• Dry for 10 min
• Resuspend in 20 µl H₂O

Result

DNA-concentration was measured at the Nanodrop c=21 ng/µl.

Re-PCR Conditions F1a.W8.A

In order to increase the product yield, F1a was amplified from the PCR fragment produced in week 7 using PCR conditions F1a.W7.A and short2 primer.

• Using hot start at 98°C

Result

Expected band: 5 Kb

Gel shows expected band at ~5 Kb.

=> Fragment was cut and gel isolated.

Generation of DelH plasmid 19-06

Ligation

Using 200 ng DNA

Using 600 ng DNA

Purification of Ligation

• Add 1 ml isopropanol
• Centrifuge 20 min full speed
• Discard supernatant
• Add 750 µl 70% ethanol
• Centrifuge 5 min full speed
• Discard supernatant
• Dry for 10 min
• Resuspend in 20 µl H₂O

Electroporation

As described in methods Electroporation of E. coli DH10β

• One Eppi with DNA of ligation with 20 µl
• Second Eppi with DNA of ligation with 50 µl (more DNA)
• Streaked on LB Amp plates. Of each electroporated aliquot, one plate with 10 µl and another with 100 µl of the cells was spread.
• Incubation ON at 37°C.

Characterization of DelH plasmid 19-06

Test Digest using SalI and PacI

Result

Expected band: 10 Kb, 8 Kb, 7 Kb
There is nothing to see on gel, most probably due to too little amounts of DNA.

Colony-PCR

• Only few colonies (~10) grew on both 100 µl plates.
• Of 8 colonies from each 100 µl plate, a colony-PCR was performed.
• Inocculation of PCR colonies into 1 ml LB Amp-Ara-XGal ON at 37°C.

Result

Expected band: 663 bp

Fig.8.3 gel of colony PCR (loaded 5 µL)
l1: 2 log ladder, l2-6: colonies 1-5 screened with PCR conditions mentioned above
l2 shows expected band

Only colony 4 (second lane) shows expected band at ~600 bp. Yet, no blue color could be detected in ON culture, even in pellet (frozen for SDS Page).

=> This means, that our ligated plasmid was successfully transformed, but lacZ is not expressed.

Will run SDS-PAGE of transformed cultures and parental stock. Also inocculate cultures using higher concentrations of arabinose and X-Gal.

Stronger Induction of lacZ Expression

• Inocculated single colonies from day before into 5 ml LB Amp-Ara-XGal using 2x amounts of arabinose and X-Gal stock solutions (maybe concentrations of stocks are too low or promoters need stronger induction).
• Incubation ON at 37°C.

Result

No lacZ expression was observed.

24-06 - 30-06-13

Characterization of DelH plasmid 19-06

SDS Page

• Using NuPAGE Novex 10% Bis-Tris precast gel from Invitrogen with MOPS running buffer

3NuPAGE Novex 3-8% Tris-Acetate precast gel would have been more suitable considering expected 600 kDa DelH, but needed Tris-Acetate SDS buffer was not available.

• Resuspended pellets in 100 µl SDS buffer (from Linda)
• Boiled for 10 min @98°C
• Ran for 90 min, 180 V
• Stained for 40 min in Coomassie Buffer (50 ml acetic acid 100% + 225 ml ddH₂O, 1.5 g Coomassie in 225 ml methanol, mix both)
• Washed in ddH₂O multiple times

Result

Fig.9.1 SDS PAGE
L1: Ladder L2: 6 µl of Konrad’s E. coli L3: 6 µl DelH colony 4 L4: 6 µl DelH colony 6 L5: 9 µl of Konrad’s E. coli L6: 9 µl DelH colony 4 L7: 9 µl DelH colony 6 L8: 15 µl of Konrad’s E. coli L9: 15 µl DelH colony 4 L10: 15 µl DelH colony 6 L11: empty L12: Ladder

None clear band at ~600 kDa visible. Maybe in highest amounts, but not reliable.

=> Probably no DelH expression in analyzed colonies.

Mini Prep

• Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9)
• Incubation ON at 37°C
• 5 ml ON cultures were mini prepared and resuspended in 50 µl

Test Restriction Digest

• 5 µl of the mini prep were test digested

• Incubation at 37°C for 1 h

Result

Expected band pattern: 5 kp, 7.3 kp, 13 kp

Fig.9.2 Gel of amplified fragments (loaded 20 µL)
l1: 2 log ladder, l2:test digested DelH fragment
l2: no visible band

No clear bands visible.

=> DNA yield from mini prep was probably too low.

DNA Concentration

DNA measurement confirmed suspicion from test digest.

=>Thus, mini prep and test digest have to be repeated

Repetion of Mini Prep

• Picked colonies from LB Amp plates into 5 ml LB Amp (colonies 4, 6 and 9) or from remaining ON culture (colony 1)
• Incubation ON at 37°C
• Colony 1 did not grow (also not on LB Amp plate), obviously lost plasmid
• Mini preped remaining 3, eluted in 35 µl ddH₂O

DNA Concentration

Test Restriction Digest

• Digested entire mini prep (using remaining from first mini for colony 1) for 2 h at37°C

Result

Expected bands: BB (7.3 Kb), DelH F2 (8 Kb), F1a + F1b (10 Kb)

Fig.9.3 miniprep resitriction digested (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1a
l2: DelH-F1a shows specific band = cut out

Unexpected bands at 3 Kb, but not desired ones.

=> Colonies do not contain desired plasmid and entire ligation has to be repeated.

Amplification of DelH F1b

PCR Conditions F1b.W9.A

• Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.4 gel of amplified DelH 1b - fragment (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1b
l2: DelH-F1b shows no band

No correct band visible.

=> Why could we not reproduce the previous amplification?

PCR Conditions F1b.W9.B

• Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.5 gel of amplified DelH-1b-fragment (loaded 20 µL)
l1: 2 log ladder, l2: DelH-F1b
l2: DelH-F1b shows unexpected band at 2.3 Kb

Again, expected band not there.

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.

PCR Conditions F1b.W9.C

• Using hot start at 98°C

Result

Expected band: 5 Kb, Loaded 1 µl of PCR

Fig.9.9 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified

Fig.9.8 gel of amplified fragments (loaded 20 µL)
l1-3:F1b, l4: 2log ladder, l5-7: BB
BB was not amplified

Fig.9.7 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 5 Kb = was cut out

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 5 Kb

Highly specific band at 5 Kb, as well as additional smaller ones.

=> Band was cut. Run gel with remaining sample for gel extraction.

PCR Conditions F1b.W9.D

• Using hot start at 98°C

Result

Expected band: DelH F1b 5 Kb

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!

Amplification of DelH F2

PCR Conditions F2.W9.A

• Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.11 gel of amplified fragments (loaded 1 µL)
l1: 2log ladder, l2:F1b, l3: F2, l4: BB
shows band at 3 Kb

Unexpected bands at 3 Kb in lanes of DelH 2

=> Why could we not reproduce the previous amplification?

PCR Conditions F2.W9.B

• Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.12 gel of amplified fragments (loaded 1 µL)
l1:2log, l2: F1b, l3: F2, l4: BB
F2 shows no specific band

Unexpected bands at 1.5 Kb, 2.2 Kb, 4 Kb and 6 Kb. Desired 8 Kb band is present but hardly visible.

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.

PCR Conditions F2.W9.C

• Using hot start at 98°C

Result

Expected band: 8 Kb

Fig.9.9 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL)
l1:2log ladder, l2: F1a, l3:F2
l3: F2 shows specific band at 8 Kb and other ones

Fig.9.8 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 19 µL)
l1:2log ladder, l2: F1a, l3:F2
l3: F2 shows specific band at 8 Kb and other ones

Fig.9.7 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l2: F1a shows specific band at 8 Kb = was cut out

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 1 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l3: F2 shows specific band at 8 Kb and other ones

F2 shows specific band at 8 Kb and additional ones.

=> Band was cut. Run gel with remaining sample for gel extraction.

PCR Conditions F2.W9.D

• Using hot start at 98°C

Result

Expected band: 8 Kb, Loaded 1 µl of PCR

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!

Amplification of Backbone pSB6A1-AraC-lacZ

PCR Conditions BB.W9.A

• Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.11 Gel of amplified fragments of DelH and BB(loaded 1 µL)
l1:2log ladder, l5: F1b, l6: F2, l7: BB
no product

No band visible, amplification failed.

=> Why could we not reproduce the previous amplification?

PCR Conditions BB.W9.B

• Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig. 9.12 Gel of amplified DelH-fragments and BB l1:2log, l2: F1b,l3:F2, l4:BB

Unexpected band at 2.2 Kb

=> Adapt PCR conditions and ask for polymerase used last time, which is the one from the lab upstairs.

PCR Conditions BB.W9.C

• Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.6 Gel of amplified DelH-fragments(F1a & F2) and Backbone (pSB6A1-AraC-lacZ) (loaded 20 µL)
l1:2log ladder, l2: F1a, l3:F2, l4: BB
l4: BB shows no expected band

Gel does not show expected fragment.

=> Make sure right template was used. Is the restricted and purified fragment suitable?

PCR Conditions BB.W9.D

• Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.9.10 gel of amplified fragments using Q5 (loaded 1 µL)
l1:2log ladder, l5: fragment 1b, l6: fragment 2, l7: BB
no product

Neither of the PCRs yield any product.

=> Therefore, Q5 is no option for us!

01-07 - 07-07-13

Amplification of DelH F1b

PCR Conditions F1b.W10.A

• Using hot start at 98°C

Result

Expected band: 5 Kb

Fig.10.1 gel of amplified fragments (loaded 1 µL of PCR)
l1: 2log ladder, l2: F1b,l3: F2, l4: BB
no bands

No correct bands visible.

=> Phusion is no option for us, not even for re-PCRs.

PCR Conditions F1b.W10.B

• Using hot start at 98°C

Result

Expected band: 5 Kb

Fig.10.2 gel of amplified DelH fragments (loaded 50 µL of PCR)
l1-2: F1b, l3: 2log,l4-5:fragment 2 of DelH, l6-7: pSB6A1
l1: F1b shows no band

=> F1b was not amplified.

Amplification of DelH F2

PCR Conditions F2.W10.A

• Using hot start at 98°C

Result

Expected band: 8 Kb, Loaded 1 µl of PCR

Fig.10.1 gel of amplified fragments (loaded 50 µL of PCR)
l1: F1b, l2: 2, l3: pSB6A1
l2: F2 shows no band

Gel does not show correct band.

=> Phusion is no option for us, not even for re-PCRs.

PCR Conditions F2.W10.B

• Using hot start at 98°C

Result

Expected band: 8 Kb, Loaded 50 µl of PCR

Fig.10.2 gel of amplified fragments (loaded 50 µL of PCR)
l1-2: F1b, l3: 2log, l4-5: 2,l6-7 pSB6A1-AraC-lacZ
l1: F1b shows no band

=> F2 was not amplified.

Amplification of Backbone

PCR Conditions BB.W10.A

• Using hot start at 98°C

Result

Expected band: 7.3 Kb, Loaded 1 µl of PCR

Fig.10.1 gel of amplified fragments (loaded 1 µL of PCR)
l1: 2log ladder, l2: F1b,l3: F2, l4: BB
no bands

No product.

=> Phusion is no option for us, not even for re-PCRs.

PCR Conditions BB.W10.B

• Using hot start at 98°C

Result

Expected band: 7.3 Kb

Fig.10.2 gel of amplified fragments (loaded 50 µL of PCR)
l1-2: F1b, l3: 2log,l4-5:fragment 2 of DelH, l6-7: pSB6A1
l1: F1b shows no band

=> BB was not amplified completely. It seems that there is no insert present or the conditions were not optimal. Repeat the PCR with other conditions.

PCR Conditions BB.W10.C

Result

Expected bands: 7.3 Kb, Loaded 50 µl of PCR

Fig.10.4 gel of amplified fragments (loaded 50 µL of PCR)
l1: F1b, l2: 2, l3: pSB6A1
specific bands at 7,3 Kb was cut out

Fig.10.3 gel of amplified fragments (loaded 50 µL of PCR)
l1: F1b, l2: 2, l3: pSB6A1
band at specific 7,3 Kb, but also unspecific ones

Expected band is there, but also unspecific ones.

=> Correct band was cut and gel extracted.

Generation of DelH Plasmid

Current Status

Restriction Digest of Fragments

• 1 h at 37°C

Isopropanol Purification

Confirmation of DNA Concentration

Fig.10.5 concentration validation (loaded 1 µL of F1a & F2 PCR)
l1: F1a, l2: F2, l3: 2log
there are tiny bands showing the expected fragment for F2, F1a shows no band

Fragment F1a is not present, all others are fine.

=> Therefore, the next steps are:

1. Measurement of concentration = 40 ng/µl

2. PCR of the fragment F1a. As template, we use the purified PCR with the concentration of 40 ng/µl.

Restriction Digest of F2 and Backbone

• 1h at 37°C

Purification of Restriction Digest

Using nucleotide removal kit (Qiagen), eluted with 30 µl ddH₂O.

Amplification of DelH F1a

PCR Conditions F1a.W10.A

Result

Expected band: 5 Kb

Fig.10.6 gel of amplified fragments (loaded 20 µL)
l1: F1a ,l2: 2log ladder,l3:F1b
no band visible

Gel does not show any bands.

=> We conclude, that the fragments we expected to be F1a cannot be amplified by PCR. Thus, they were not amplified in the PCR used as template.

PCR Conditions F1a.W10.B

Result

See Confirmation of DNA Concentration

Amplification of DelH F1b

PCR Conditions F1b.W10.C

Result

Expected band: 5 Kb

Fig.10.6 gel of amplified fragments (loaded 20 µL)
l1: F1a ,l2: 2log ladder,l3:F1b
no band visible

There is no band visible.

=> We conclude, that the fragments we expected to be F1b cannot be amplified by PCR. Thus, they were not amplified in the PCR used as template.

Generation of DelH Plasmid

Confirmation of DNA Concentration

Fig.10.7 gel of fragments (amplified by PCR) (loaded 20 µL)
l1: F1a, l2: F1a, l3: F1b (digested (D) & purified (P)), l4: F1b (D+P), l5: F2(D+P), l6: F2(D+P), l7: F2, l8: BB(D+P), l9: BB, l10: BB(D+P), l11: 2log
lane 2, 3, 4, 5 had no band

DelH F1a (40 ng/µl) was the fragment we used for the first ligation. It is not visible, thus, there was not enough little DNA. Since the screening primer binds to this part of DelH, we could not identify any positive clone.

=> The negative samples (with too little DNA concentration) are discarded.

Amplification of DelH F1a

PCR Conditions F1a.W10.C

Result

Fragment was gel extracted.

Amplification of DelH F1b

PCR Conditions F1b.W10.D

Result

Expected band: 5 Kb

Fig.10.9 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

No bands visible.

=> Fragment F1b could not be amplified from neither genomic DNA nor PCR fragment. Amplification will be repeated using DMSO in the mix.

PCR Conditions F1b.W10.E

• Using hot start 98°C

Result

Expected band: 5 Kb

Fig.10.10 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

Only PCR of fragment 19 ng/µl + DMSO was successful. Maybe there is another pale band at the second PCR of DelH F1b.

=> Repeat both PCRs.

Result

PCR using conditions F1b.W10.E was repeated.
Expected band: 5 Kb,Entire PCR mix was loaded.

Fig.10.12 gel of amplified fragments (loaded 20 µL)
l1/2: entire F1b, l3/4: older PCR of F1b, l5: 2log ladder, l6/7 BB
excised band of lane 6/7

Fig.10.11 gel of amplified fragments (loaded 20 µL)
l1/2: entire F1b, l3/4: older PCR of F1b, l5: 2log ladder, l6/7 BB
excised band of lane 6/7

PCR did not result in any fragments. Interestingly, both older PRCs resulted in a fragment at ~5 Kb and other lines, but the fragment from the genomic DNA is slightly larger.

=> Both fragments were cut and gel isolated, check correct length of F1b!

=> Perform PCR from the PCR fragment obtained from the genomic DNA.

Amplification of DelH F2

PCR Conditions F2.W10.C

• 2x 50 µl

Result

Expected band: 8 Kb

Fig.10.9 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

Fragment 2 was successfully amplified.

=> It was cut and gel purified.

Preparation of new Template DNA

• D. acidovorans glycerol stock was streaked onto LB plates and incubated ON at 37°C.
• D. acidovorans did not grow, since LB media is not appropriate for them. Prepare new ACM media and plates and streak D. acidovorans.

Amplification of Backbone

PCR Conditions BB.W10.D

• 2x 50 µl

Result

Expected band: 7.3 Kb

Fig.10.13 gel of amplified fragments (loaded 20 µL)
lane 1 2log ladder, lane 2 fragment 1, lane 3 fragment 2
excised band of lane 3; lane 2 had no band

No band visible.

=> Repeat using DMSO.

PCR Conditions BB.W10.E