Team:Exeter/lab book/1gp/wk4

Operon Construction  |  Glycobase

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• In duplicate, 1.5mL of overnight BL21(DE3) broth culture was transferred to a fresh, labelled 50mL Falcon tube and pelleted by centrifuging for 2 minutes at 13’000 x g. The supernatant culture medium was removed completely and discarded.

• The pellet was re-suspended thoroughly in 180µL lysis solution T and 20µL RNase A solution was added. The total solution was mixed and incubated for 2 minutes at 22oC.

• 20µL Proteinase K was added to the solution and then mixed. This was then incubated for 30 minutes at 55oC.

• 200µL lysis solution C was added and the solution was vortexed thoroughly for about 15 seconds and then incubated again at 55oC for 10 minutes.

• Whilst this was incubating, 500µL column preparation solution was added to a pre-assembled GenElute miniprep binding column that was seated in a 2mL collection tube. This was centrifuged at 12’000 x g for 1 minute. Afterwards, the eluate was discarded.

• When incubation of extracted genomic DNA of BL21(DE3) was complete, 200µL ethanol (100%) was added to the lysate and mixed thoroughly by vortexing for 5-10 seconds.

• All the lysate-ethanol solution was transferred to the GenElute miniprep binding column, being careful to not pipette too hard as to shear the genomic DNA. The binding column with the lysate-ethanol solution was centrifuged at 6’500 x g for 1 minute. After this, the collection tube was discarded that contained the eluate and the binding column was placed in a new 2mL collection tube.

• 500µL wash solution 1 was added to the column and centrifuged for 1 minute at 6’500 x g. The 2mL collection tube after centrifugation was discarded and the binding column was added to a new 2mL collection tube. 500µL wash solution containing ethanol was transferred to the binding column and centrifuged for 3 minutes at 13’000 x g to dry the column. The column was centrifuged for another minute so all ethanol was removed from the binding column. The collection tube containing eluate (including ethanol) was discarded and the binding column was placed in a new 2mL collection tube.

• 200µL elution solution was pipetted directly onto the center of the binding column and left to incubate at 22oC for 5 minutes. After this, the column was centrifuged for 1 minute at 6500 x g to elute the genomic DNA. This was repeated again to elute as much DNA as possible using the same 2mL collection tube.

• The 2mL collection tube containing genomic DNA was stored on ice (2-8oC), and 1.5µL was used to measure DNA concentration on the Nanodropping Machine.

• To PCR amplify WbbC from the genomic DNA, a two master-mixes were made up containing: 10µL 5ng/µL genomic DNA (starting concentration = 7.8ng/µL for the first PCR tube and 6.6ng/µL for the second PCR tube), 1µL of dNTP’s, 1µL WbbC forward primer (at 100pmol/µL), 1µL WbbC reverse primer (at 100pmol/µL), 10µL 5x PCR buffer, 1µL Phusion polymerase and 26µL MilliQ H2O to make a total volume of 50µL.

• Once this master-mix was made up, the PCR tube containing these reagents was put in a PCR machine that was programmed for two-step PCR (using Phusion). Because of the high TM of the forward and reverse primers for WbbC (roughly 75oC), two-step PCR using extension at 72oC was possible. The machine was programmed as: 98oC for 30 seconds, 98oC for 10 seconds and then 72oC for 2 seconds (this was repeated for 30 cycles), and then finally 72oC for 5 minutes.

• As WbbC was being PCR amplified, an agarose gel for gel electrophoresis was made to verify whether WbbC was amplified. This involved adding 50mL 1x TAE buffer to 0.5g agarose and then microwaving until completely dissolved.

• 1µL ethidium bromide (EtBr) was added to the hot dissolved agarose gel and mixed thoroughly. The entire solution was poured into a gel electrophoresis plate with a well comb inserted. Once left to set for around 15 minutes, the well comb was lifted out.

• When PCR amplification of WbbC was finished, 5µL PCR mix was added to 1µL loading buffer to make a 5x dilution. 5µL of DNA hyperladder was also added to another tube containing 1µL loading buffer (again, to make a 5x dilution).

• Both DNA hyperladder and PCR mix were added to different wells and then TAE buffer was added to the gel electrophoresis plate to submerge the agarose gel and therefore the two samples.

• Gel electrophoresis was then conducted at 200V for 15 minutes.

• After UV illumination, no WbbC products in lanes 2 and 3 were observed. It was discovered that the reverse primer was incorrect and had actually amplified a shorter section of WbbC, rather than the whole gene. Thus the correct reverse primer was ordered.

• Meanwhile, transformed competent E.coli containing recombinant pBAD/AraC promoter plasmids were picked from the 100µL spread plate and inoculated in LB broth (See: Week 30th July-03rd August 2012, Operon Construction: Mary Beton).

Tuesday 31st July (15.00) – IDT Re-suspension and Transformation

• The synthesised gene WbbC (with a point-mutation) was re-suspended using IDTE buffer (40µL of 10mM Tris, 0.1mM EDTA), held between pH 7.5-8.0, into a fresh Eppendorf tubes, to reach an approximate concentration of 50ng/µL stock solution.

• The tube was vortexed for 20 seconds, left to incubate at room temperature for 30 minutes, and then centrifuged for 1 minute.

• To dilute WbbC for the transformation procedure, 2µL of re-suspended WbbC was added to MilliQ H2O (999µL) to reach an approximate concentration of 0.1ng/µL in a fresh, labelled Eppendorf. The tube was centrifuged briefly to spin down any liquid.

• After this, 2µL of diluted WbbC was pipetted gently into 50µL Top10 E.coli competent cells (Invitrogen), making sure not to mix too rigorously.

• Competent cells were then incubated on ice for 30 minutes.

• They were then heat-shocked for exactly 30 seconds in a 42oC water bath, making sure not to mix or shake.

• Afterwards, they were quickly placed on ice for 2 minutes.

• Pre-warmed SOC medium (250µL) was added and then the Eppendorf tube containing recombinant WbbC plasmids was secured in a shaking incubator and incubated at 36.8oC for 1 hour at 220rpm.

• Whilst incubating, two LB agar plates were made-up (25µL LB agar in each). One would be used for spreading 20µL of transformed E.coli competent cells and the other for 100µL of transformed E.coli competent cells, so that we would be able to pick out enough isolated colonies. 50µL ampicillin was added to 50mL of LB agar to create a 1000-fold dilution.

• After the transformed E.coli competent cells had finished incubating for an hour, 20 or 100µL of the transformants were spread plated onto respective ampicillin-containing LB agar plates.

• Remaining transformation mix was stored at 4oC and the two inoculated LB agar plates were inverted and placed in an incubator at 37oC overnight.