Silver:Restriction digest protocol

Solutions/reagents:  restriction enzyme1  (20 units/µl)  restriction enzyme2  (20 units/µl) 10X restriction buffer10X BSAPCR product DNAdistilled H2O <a name="BioBrick vector">BioBrick vector  (BBa_V0002 or BBa_V0100) </a></li> <a name="CIP">CIP  (10 units/µl) </a></li></ul> Parameters: V - Volume of PCR product DNA obtained (µl).</li>C - Concentration of BioBrick vector (µg/µl).</li></ul> Equipment: Incubator</li>Reaction tubes</li></ul> Steps: <ol>  Digestion of PCR product  Use the following table as a checklist for preparing the reaction in reaction tube (1): <font color = "#800517">Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume. </li>  Incubate at <font color=#357EC7>37°C  for at least <font color=#357EC7>1 hr . <font color = "#800517">Incubation overnight is better. </li> <font color = "#800517">Purify digested insert using PCR product purification kit. </li>   Digestion of BioBrick vector: Recommended procedure  Use the following table as a checklist for preparing the reaction in reaction tube (2):  <font color = "#800517">Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume. </li>  Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>12 hrs (overnight). </li> Measure out <font color=#357EC7>0.1 µl  of <a href="#CIP" ><font color=#357EC7>CIP </a> into reaction tube (2). Incubate at <font color=#357EC7>37°C  for <font color=#357EC7>1 hr . </li> </ol> TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 14 hrs, 0 mins