Fatih KocabaÅŸ

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

94^oC       1 min

94^oC       30 sec

58^oC       1 min          30 cycles

72^oC       1.5 min

72^oC       10 min

4^oC         ∞

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 50^oC 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 37^oC 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 37^oC 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 25^oC 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 25^oC 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: 42^oC bath, 45 seconds

·         Immediately incubate on ice for 2 min

·         Add 900 ul of SOC medium or LB medium

·         Recovery of cells: Incubate at 37^oC 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 37^oC, O/N, plates are facing down.