PCR, real-time PCR, primers, column and SPRI clean-up of reactions
Preparation of PCR primer stocks and working solutions
stocks
spin freezedried stocks, 1min full speed
add sterile H2O (molecular biology grade) to a final concentration of 100µM
working solutions
485µl sterile H2O (molecular biology grade)
+ 15µl primer stock
--> 3µM
DNA column purification (Qiagen PCR clean-up / Qiaquick Gel Extraction)
Loading
mix reaction + 5Vol PBI buffer in Epi
place column in collection tube
load on column
spin 1min, full speed, RT
discard floughthrough
alternatively for gel extraction of DNA bands
cut bands on blue light table (DeLong lab), or on UV Transilluminator (Thompson lab) with clean razor blade
transfer into Epi
weigh Epis (~1g) and gel slices
+3Vol/weight (300µl/100mg) GC buffer
incubate tubes at 50˚C, 10min, vortex gently every 2min
+ 1 Vol/weight (100µl/100mg) Isopropanol (improves yield especially for fragments below 500bp or above 4kb)
mix by inverting tube
place column in collection tube
load on column (if volume to big for column, then load, spin, discard flowthrough, and load rest)
spin 1min, full speed, RT
discard floughthrough
Washing
+750µl PE buffer (seal bottle tight to avoid EtOH evaporation)
spin 1min, full speed, RT
discard floughthrough
place column back in empty collection tube
Drying
spin (to dry) 30sec, full speed, RT
turn column in collection tube by 180˚
spin (to dry) 1min, full speed, RT
discard floughthrough and collection tube
place column in new Epi
dry open column under laminar air flow for 2min
Elution
+35-50µl EB or sterile H2O (molecular biology grade)
incubate at least for 5min
spin (to elute DNA) 30sec, full speed, RT
turn column in Epi by 180˚
spin (to elute DNA) 1min, full speed, RT
discard column and store Epi with DNA
SPRI clean up and primer/dimer removal (Agencourt AMPure XP beads)
Preparations:
adjust PCR reaction to 50µl with EB
vortex SPRI beads 1600rpm, 10sec
aliquot 45µl of beads into one 1.5ml tube per library
Binding to beads
add 50µl PCR reaction to beads
mix by pipetting/vortex 1600rpm
incubate for 5-7min
separate on magnet for 2min
remove and discard SN while tube stays on magnet
Wash - removal of salts, enzymes and low molecular weight DNA
wash beads carefully twice with 70% EtOH while tube stays on magnet (do not disturb bead pellet)
incubate for 30sec
remove all SN
repeat
Dry
air dry on magnet for 15min
Elution
remove tube from magnet, add 20µl EB
vortex 1600rpm, 10sec
incubate at RT for 5min
separate on magnet for 2min
collect SN and transfer into new 1.5ml tube
Quantitative real-time PCR
Mastermix
for 200µl (8x) reaction:
10.525µl H2O
5µl 5x Phusion Pol buffer
0.5µl dNTP mix 10mM
3.3+3.3µl primers, 3µM
2µl template (try different 10 fold dilutions)
0.125µl SYBR Green I
0.25µl Pol (Phusion)
prepare reaction in PCR tubes (or 96well plate) with optical covers that fit the utilized
real-time PCR
cycle at 1) 98˚C, 20''
2) 98˚C, 15''
3) specific Annealing Temp˚C, 20''
4) 72˚C, 20'' (for fragments shorther than 1kb)
5) go back to step 2 45x
always use at least 3 replicates per sample
always include 3 replicates of a non-template (H2O) control