Team:ETH Zurich/Wet Lab/Protocol

Introduction of GFP/RFP into MG1655/MD42 E. coli strains

using the Lambda Red System

Subcloning of GFP/RFP into pCK01 followed by PCR amplification

Day 1: Friday, July 11

• digest p18GFP/BB_J14461RFP plasmids and pCK01 2.5 h

  • 25 µL GFP construct/RFP construct/pCK01 plasmid

1 µL EcoRI

1 µL PstI

3 µL EcoRI buffer

→ 2 h at 37°C

→ pCK01 digest was heat-inactivated for 20 min at 80°C

• run gel to confirm digest 1 h

  • 0.8 % agarose gel:

lane 1 (large!) + 2: 26 µL RFP digest + 6 µL loading buffer (LB)

lane 3: 4 µL RFP digest + 2 µL LB

lane 4: 2.5 µL λ marker + 2 µL LB

lane 5 (large!): 26 µL GFP digest + 6 µL LB

lane 6: 4 µL GFP digest + 2 µL LB

→ run at 90 V for ca. 1 h

→ lanes 3/4 and lane 6 cut out and put in EtBr for ca. 30 min

pics (4 s exposure) saved under GFP_RFP_restr

• cut out GFP/RFP bands, recover DNA 1 h

  • RFP showed 2 bands: one at ca. 1000 bp and one higher

(ca. 1000 bp + higher expected)

• GFP showed 3 bands: one at ca. 1000 bp, one higher and one lower

(ca. 500, 1000 and 2200 bp expected)

→ bands at 1000 bp were cut out (ca. 250 mg)

• gel extraction was performed according to protocol:

750 µL of buffer QG

250 µL isopropanol

elution with 30 µL EB buffer (wait 1 min before elution!)

• ligate recovered DNA with digested pCK01 O/N

  • 3 µL pCK01 vector

1 µL T4 buffer

0.5 µL T4

5.5 µL GFP insert/RFP insert/ water (control)

                        → spin down and put to 4°C O/N

Day 2: Saturday, July 12 (5 pm)

• transform ligations (3 x) and GFP/RFP/pCK01 plasmids 2 h

(to get backups): 6 transformations!

• 3 ligations transformed:

10 µL ligation (GFP/RFP/control)

40 µL competent cells

• 3 backup plasmids transformed:

2 µL p18GFP/BB_J14461 RFP/pCK01 plasmids

40 µL competent cells

→ 20 min on ice

→ 90 sec at 42°C

→ 90 sec on ice

→ 200 µL CVM added

→ 1 h recovery at 37°C and 220 rpm

• plate out transformations (6 x) and incubate at 37°C O/N

• 3 ligations (GFP-pCK01, RFP-pCK01, control) plated on

LB + chloramphenicol (cat) + x-gal agar plates

• pCK01 plated on LB + chloramphenicol agar plates, p18GFP and BB_J14461 RFP plated on LB + ampicillin agar plates

→ put to 37°C O/N

Day 3: Sunday, July 13 (5 pm)

• pick colonies and inoculate over night cultures: O/N

1 colony of GFP/RFP/pCK01 and lambda red

+ 2 x 5 of GFP-pCK01/RFP-pCK01 → 14 cultures

• one GFP/RFP/lambda red colony was inoculated in 15 mL-Falcon

tubes containing 5 mL LB medium + ampicillin

• 5 colonies of GFP-pCK01/RFP-pCK01 were inoculated in 5 mL

LB medium + chloramphenicol

→ put to 37°C O/N

Day 4: Monday, July 14 (9 am)

• do mini preps of GFP-pCK01, RFP-pCK01 (3 x each) and GFP/RFP/pCK01/lambda red plasmids (10 mini preps):

  • eluted in 50 µL EB buffer after 1 min incubation at RT

• digest and gel to check cloning:

  • GFP/RFP plasmids digested:

                    2 µL DNA

                    1 µL EcoRI buffer

                    0.5 µL EcoRI

                    0.5 µL PstI

                    6 µL water

• GFP-pCK01/RFP-pCK01/pCK01 plasmids digested:

                    8 µL DNA

                    1 µL EcoRI buffer

                    0.5 µL EcoRI

                    0.5 µL PstI

                    → put to 37°C for 2 h

• run on 0.8 % gel:

                    add 2 µL LB

                    load 10 µL of each

                    run at 100 V

                    take pictures

                    → hardly any DNA visible

• Inoculation of an additional 3 colonies of GFP-pCK01 and RFP-pCK01
  • inoculated in 5 mL chloramphenicol containing LB medium
  • incubated at 37°C O/N

Day 5: Tuesday, July 15

• GFP-pCK01, RFP-pCK01 (3 x each) and GFP/RFP/pCK01/lambda red plasmids were run on 0.8% gel without digestion

          → double bands (several colonies picked?!)

• Mini prep of 2 x 3 GFP-pCK01/RFP-pCK01 cultures
  • mini preps (2 x 2) were performed and run on 0.8% gel

                    → 3/4 positive

Day 6: Wednesday, July 16

• do PCR of GFP-pCK01 and RFP-pCK01 constructs using the ordered primers
  • primers were dissolved in xxx µL/xxx µL ddH2O (forward/reverse)
  • Master-Mix (MM):

                    40 µL 5 x Taq buffer

                    32 µL 25 mM MgCl2

                    2 µL ATP

                    2 µL GTP

                    2 µL CTP

                    2 µL TTP    

                    8 µL forward primer (100 µM)

                    8 µL reverse primer (100 µM)

                    8 µL Taq polymerase

                    272 µL ddH2O

• 3 different amounts of GFP-pCK01 (no. 2, 15.07.08)/RFP-pCK01 (no. 1, 15.07.08) template were used:

                    49 µL MM + 1 µL template

                    48 µL MM + 2 µL template

                    46 µL MM + 4 µL template

                    → 6 PCRs

• PCR program:

                    5 min 95°C

                    35 x

                    1:50 min 95°C

                    1:50 min 50°C

                    2 min 72°C

                    5 min 72°C

                    4°C after finish; running time: ca. 4:30 h

• check and purify PCR product on gel
  • 10 µL LB added to all 6 PCR products
  • 6 µL loaded for checking
  • rest (ca. 50 µL) loaded in large pockets for extraction
  • 6 µL lambda marker loaded (3 µL marker + 3 µL LB)
  • run at 100 V on 0.8% gel

• repitition of PCR with changed protocol
  •  2.5 µL high fidelity buffer (with MgCl2, no.2)

• program:

• 0.8% gel to check and extract PCR product
  • 5 µL LB was added to each
  • run at 100 V

• gel extraction
  • gel extraction was performed using 1100 µL buffer QG and 350 µL isopropanol                 
  • samples were eluted with 30 µL EB buffer (wait 1 min before centrifugation!)

• run extracted PCR products on 0.8 % gel to check extraction and size
  • Agarose gel electrophoresis of PCR products (2µl 100 bp Fermentas DNA Marker; 3µl PCR product GFP 0.5, GFP 1, RFP 0.5, RFP 1)
  • PCR product is larger than 1kb, yield exceeds 30ng/ul

Day 1: Thursday, July 10

plate out glycerol stocks of MG1655 and MD42 done

• scrape off some of icy glycerol stock and plate on LB agar

→ incubate at 37°C O/N

Day 2: Friday, July 11

• put colonies to 4°C

  • colonies have grown and are ready to be picked!

Day 3: Monday, July 14

• prepare competent MG1655/MDS42 cells

  •  2 colonies of MG1655 and MDS42 each inoculated in 5 mL LB medium (5 pm)

  • incubated at 37°C O/N
     

Day 4: Tuesday, July 15

• prepare competent MG1655/MDS42 cells (continued)
  • 50 mL LB medium were inoculated with 500 µL over night cultures and grown to OD(600) 0.488 (MG1655) and 0.503 (MDS42)

                    (MG1655 grew faster and was stored on ice while MDS42 was still growing)

• cultures were transfered to 50 mL Falcon tube (on ice!) and spun down for 10 min at 2500 rpm and 4°C
• discard supernatant
• pellet was resuspended in 50 mL buffer 1 (pre-cooled!)
• store on ice for 90 min
• spin 10 min at 2500 rpm and 4°C
• discard supernatant
• resuspend pellet in 2 mL ice cold buffer 2
• 200 µL each were filled in 1.5 mL Eppis and stored at -80°C

• transform competent MG1655/MDS42 cells with lambda red constructs (pKD46)
  • 2 µL pKD46 were transformed into 50 µL of competent MG1655/MDS42 according to the protocol
  • heat shock at 42°C
  • 200 µL CVM added
  • recovery at 30°C
  • plated on ampicillin containing agar plates at 30°C (!) O/N

 Day 5: Wednesday, July 16

• prepare competent lambda red MG1655/MD42 pKD46 cells

  • inoculate 2 colonies of each MG1655/MDS42 pKD46 in 5 mL ampicillin containing LB medium

  • incubate at 30°C O/N at 200 rpm

Day 6: Thursday, July 17

• prepare competent lambda red MG1655/MD42 pKD46 cells (continued)
  • protocol as described on day 4 (with MG1655/MD42 pKD46 cells!)
  • grown to OD(600) of 0.458 (MG1655)/0.536 (MDS42)

• cells were stored on ice for 20 min before 1st centrifugation
• aliquoted in 2 x 11 Eppis a 200 µL and stored at -80°C

• transform MG1655/MD42 pKD46 cells with GFP-pCK01/RFP-pCK01 PCR products (see above)

  • temperature shock transformation of MG1655/MD42 pKD46 cells with GFP-pCK01/RFP-pCK01 PCR products according to protocol: