Troubleshooting - Ibi IB44000 Operation Manual

DNA / RNA Semi-Dry Transfers
DNA Solutions:
• Solution 1
• Solution 2
• Solution 3
• 37% formaldehyde stock solution
• 1.8% formaldehyde stock solution – 5ml of 37% stock into 100ml of DI water
• 6.5% formaldehyde stock solution - 1.75ml of 37% stock into 10ml of DI water.
Requirements for Electro-Transblotting DNA and RNA
Method
DNA/RNA from
Acrylamide or
agarose gels
RNA from Agarose
Gel thickness and buffer solutions will affect run time.
Problem
Poor electrophoretic
Transfer of DNA or RNA
Recommended Solutions for
Denaturing solution 0.5M NaOF, 1.5M NaCl
Neutralizing solution 1.0M TRIS-Hcl/pH 8.0
1.5M NaCl
1X TBE 0.089M TRIS
0.089M Boric Acid
0.002M EDTA, pH 8.3
10X TBE 108gm TRIS base
55gm Boric acid
40ml 0.5 EDTA, pH 8.0
1X MOPS 4.186gm MOPS (20mM MOPS)
41gm sodium acetate (5mM Sodium acetate)
2ml of 0.5M EDTA pH 8.0 (1mM EDTA)
Dissolve the MOPS, sodium acetate and EDTA into 880ml of DI water.
Recommended Current
1.5 – 3.5mA/cm
2
2.0 – 3.5mA/cm
2

Troubleshooting

Cause
Buffer may be too
concentrated causing
gel to carry too much
much current.
Power conditions during
transfer may have changed
If the voltage is no set high
enough the current draw will
drop below optimal range for
good electrophoretic transfer
Gel Size
9 x 9cm
16 x 16cm
9 x 9cm
16 x 16cm
Reduce buffer concentration
Optimum transfer of plasmid,
vector and PCR DNA appear
to occur when amps are set
between 3 – 3.55mA/cm
to 15 minutes. Optimum Transfer of RNA
is 2.5 to 3mA/cm
Recommended Time
30 – 60 minutes
1 hour
30 – 40 minutes
30 – 40 minutes
Solution
to 0.5X to reduce current
Set Voltage higher
for 10
2
for 30 - 40 minutes.
2