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Research Training METHODS (In silico characterisation (Prosite (Scan SS,…
Research Training METHODS
In silico characterisation
Prosite
Scan SS
Searched for motifs specific to GTPase and OBG/YchF family
BLAST
Searched SS genome
Used 4 different protein searches
InterPro
Domains identified
OBG and TSG-like domains
TMpred, Jpred4 and ProtParam
Predictions of various structures, parameters and TM
MSA
Sequence alignment generated, highlights key motifs
Phyre
Template = YchF GTP-binding protein
Structure visualised V SIMILAR TO YCHF (but this was template...)
Transforming Alpha and Miniprep
Transformation
Alpha Select Gold Efficiency competent cells
25uL cells + 2uL InFusion reaction ON ICE
Heat shock for 30S AT 42c
200uL SOC (Media Service)
shake 299rpm for 40 mins
150uL onto AGAR PLATES (carb + IPTG + XGAL)
Grow Up
Cells grew up, lots of WHITE colonies
5mL of LB + carb
Picked 4 white colonies to grow up overnight (200 rpm, 37C)
Miniprep
Spin and pellet (15m, 4200rpm, 4C)
QIAprep miniprep kit
Checked correct insert on a 1% agarose
Transforming Expression
Transformation
BL21 (DE3) E Coli Expression Cells
25uL cells + 1 uL DNA, 30mins on ice
250uL SOC, shake 1 hr 200rpm
90s heat shock at 42C
150uL of cells onto agar plates (+Carb)
Expression
Picked colonies and put in tube of LB and carb, shake overnight at 37C 200rpm
Innoculate 1mL of overnight with LB + carb
Spectrophotometrically measure cell growth at 600nm (grow up)
IPTG added to induce expression, left at 27C overnight to express
Cells pelleted and resuspended in 1mL resusp. buffer, kept at -20C
Protein purification
Lysis
Lysozyme, DNase, TritonX, MgCl2
Vortex breifly and often 30m
Centrifuge and extract SOLUBLE FRACTION
Purifcation
His SpinTrap OR HisPur Ni-NTA Spin columns??
Visualised with Coomassie, rocker destain
Ran Mini-PROTEAN Precast Gels (SDSPAGE) for 25m 240mV
Dialysis
Pur-a-Lyzer mini dialysis kit
2L of 20mM TRIS, 150mM NaCl, 1mM DTT, 50% glycerol
Concentration
Measure A at 250-350nm
10uL sample + 90uL dialysis buffer
Quartz cuvette
Datastream
ATPase assay
All tubes
RB, PEP, PK/LDH, ATP
Quartz cuvette
100uL
Measuring ATP hydrolysis indirectly, NADH loss (340 nm)
Positive control
100nM ATPase, Rad54
Ran for 90s
1uM ssDNA
Negative controls
7.7uL of buffer
5uM poly-U RNA
Ran for 30 min
SS-OGY
1uM protein
5uM polyU RNA
Ran for 30m
SS-OGY K
50mM KCl
Ran for 30m
Amplifying, Extracting and inserting DNA into vector
PCR
Genome compiler design primers
Dissolved primers
Extension time calculated, Tm calculator gave Annealing temp
PCR programme and content
Linearised vector
pOPINF vector + HindIII + KpnI + Cut Smart Buffer
Incubated for 1 hr 37C
Check on 1% agarose gel
Gel and extraction
Ran 1% agarose with SYBR green
1st - check correct size band etc
2nd (thick comb) cut out band
QIAquick Gel Extraction Kit
In-Fusion Cloning Reaction
Estimated concentrations from agarose (against ladder)
sample = 20ng/uL, vector = 320ng/uL
need double [insert] compared to [vector]
0.5 uL vector, 3.9 uL insert
make up reaction mix, 50C for 15m then on ice