miRNA Cloning Protocol-FK 8/8/8
PRIMER DESIGN For BamHI AND XhoI RE SITES
· Add BamHI and XhoI Restriction enzyme sites to beginning of forward and reverse primers respectively
· Include additional 3-6 nucleotides before restriction sites.
PCR rxn
Takara LA Taq polymerase rxn Volume (ul)
10X PCR buffer (Takara,Mg2+) 5 ul
dNTPs 2.5 mM 8 ul
Forward Primer (1ug/ul) 1ul
Reverse Primer (1ug/ul) 1ul
DNA template containing your miRNA 1 ul
Takara LA Taq Polymerase (5 U/ul) 1 ul
dH2O-up to 50 ul 33 ul
Total-------------------------------------------- 50 ul
Cycling conditions
94oC 1 min
94oC 30 sec
58oC 1 min 30 cycles
72oC 1.5 min
72oC 10 min
4oC ∞
AGAROSE GEL ELECTROPHORESIS
1% agarose gel preparation
· Weight 1.7 mg of agarose and dissolve in 1xTBE buffer by using microwave for 2-3 min.
· Add 7.5 ul of EtBr and pour it.
Sample loading
· Add 5 ul of 10X loading into PCR rxns
· Mix well and load into wells
· Run at 150V for 30-45 min
· Cut correct bands over UV Transilluminator and put them into 1.5 tubes
GEL PURIFICATION; (FOLLOW QIAGEN GEL EXTRACTION KIT PROTOCOL)
· Weight an empty 1.5 ml centrifuge tube and tare
· Weight tubes with gels
· Dissolve Gel: Add 3X of GC buffer, dissolve gel completely by vortexing and incubating in 50oC water bath
· Bind DNA to column: Add yellow solution into gel purification columns, and centrifuge for 1 min at max speed, discard flow-through
· Remove Gel: Add another 500 ul of GC buffer to columns to remove gel completely, centrifuge 1 min at max speed, discard flow-through
· Wash column: add 750 ul PE buffer to column and centrifuge for 1 min at max speed, discard flow-through
· Remove extra PE: Centrifuge 1 more min at max speed and discard flow-through
· Elute DNA: Put columns into a new tube and add 50 ul Elution Buffer (EB). Incubate for 1 min and centrifuge for 1 min at max speed.
DIGESTION OF VECTOR AND PCR PRODUCTS
Double Digestion Rxns
Vector (plenty/v5) Amount (ul)
Sample 10ul
10x NEB Buffer 2 5 ul
10X BSA 5 ul
BamHI 1 ul
XhoI 1 ul
dH2O---up to 50 ul 28 ul
Total------------------------- 50 ul
Incubate at 37oC for 3 hrs
miRNA PCR product
PCR product 20ul
10x NEB Buffer 2 5 ul
10X BSA 5 ul
BamHI 1 ul
XhoI 1 ul
dH2O---up to 50 ul 18 ul
Total------------------------- 50 ul
Incubate at 37oC for 3 hrs
Then do Agarose gel electrophoresis and Gel Purification of double digested vector and miRNA product.
LIGATION OF MIRNAS INTO THE VECTOR
Ligation rxn mount (ul)
miRNA digested PCR product 9 ul
BamHI/XhoI cut Vector(Plenti/v5) 1 ul
5X Ligation buffer 6 ul
T4 DNA Ligase (1 ul/15 ul rxn) 2 ul
dH2O---up to 30 ul 12 ul
Total---------------------------------------------- 30 ul
Incubate at 25oC for 1 hr,and then 4 oC ∞
(-)Control Ligation rxn Amount (ul)
dH2O instead of miRNA 9 ul
BamHI/XhoI cut Vector(Plenti/v5) 1 ul
5X Ligation buffer 6 ul
T4 DNA Ligase (1 ul/15 ul rxn) 2 ul
dH2O---up to 30 ul 12 ul
Total---------------------------------------------- 30 ul
Incubate at 25oC for 1 hr,and then 4 oC ∞
TRANSFORMATION
· Thaw competent cells (50-70 ul for each miRNA)
· Pre-Chill tubes on ice
· Add 5 ul into 70 ul of cells
· Incubate on ice 10-30 min
· Heat-Shock: 42oC bath, 45 seconds
· Immediately incubate on ice for 2 min
· Add 900 ul of SOC medium or LB medium
· Recovery of cells: Incubate at 37oC for 30-90 min constant shaking
· Pour 100 ul onto Amp/LB agar plate
· Centrifuge remaining medium and discard 700 ul of supernatant, then dissolve pellet and pour 200 ul on to another plate. Spread evenly using a spreader.
· Incubate at 37oC, O/N, plates are facing down.