RNase I Treatment

> Version Number: 1.2
>
> Production Start Date: 10/30/06
>
> Version 1.2 Date: 1/17/07
>
> Author(s): Eileen Dalin
>
> Reviewed/Revised by: Name(s)

# Summary

RNase I treatment is used for the removal of RNA from genomic DNA
samples. RNase I converts single stranded RNA to mononucleoside
3'monophosphates. If the genomic DNA sample contains RNA it can cause
problems with DNA cloning.

# Materials & Reagents

  ------------------------------------------------ -------------------------- -----------------------
  **[Materials/Reagents/Equipment]{.underline}**   **[Vendor]{.underline}**   **[Stock
                                                                              Number]{.underline}**

  *[Disposables]{.underline}*                                                 

  1.5ml Microcentrifuge tubes                                                 

                                                                              

  *[Reagents]{.underline}*                                                    

  RNase I                                          Lucigen                    30104-1,2

  3M Sodim Acetate                                 Ambion                     

  100% Ethanol                                                                

  GenePure LE Agarose (Generates)                  ISC BioExpress             E-3120-500

  50X TAE Buffer                                   Invitrogen                 24710-030

  TE Buffer pH8.0                                  Ambion                     9849

                                                                              

  *[Equipment]{.underline}*                                                   

  Centrifuge 4^0^C                                                            

  Heating block 70^0^C                                                        

  Heating block 37^0^C                                                        

  Gel Box                                                                     

                                                                              
  ------------------------------------------------ -------------------------- -----------------------

# Procedure

**1. [RNase I Treatment]{.underline}**

1.  Set-up the following reaction in a 1.5ml microcentrifuge tube
    (multiple reactions can be done in different tubes):

> Note: RNase I - 10U/ul, one unit digests 100 ng of RNA per second
>
> [1x]{.underline}
>
> DNA (in H~2~0) 170ul
>
> 10X RNase I buffer 20ul
>
> RNase I [10ul]{.underline}
>
> 200ul

2.  Mix & Spin down.

3.  Incubate tube @ 37^0^C for 1 hr.

> ***Checkpoint: Check a small aliquot (5ul) on an agarose gel with a no
> treatment control. Run gel 10-15 min. If there is only a trace of RNA,
> proceed with next step, heat inactivation. If large amounts of RNA are
> still present, add another 10ul of RNase I and repeat the
> incubation.***

4.  Heat inactivate enzyme at 70^0^C for 15 min.

5.  Place tube on ice to cool.

**2. [Ethanol Precipitation]{.underline}**

1.  Add 1/10 volume of 3M Sodium Acetate to your sample.

2.  Add 2.5 volumes of 100% ethanol.

3.  Mix and spin down sample.

4.  Place at -80^0^C for 30 min (-20^0^C overnight).

5.  Spin sample at 4^0^C for 20 min to pellet DNA.

6.  Carefully, pour off supernatant.

7.  Wash pellet with 70^0^C ethanol (cold).

8.  Spin sample at 4^0^C for 3-5 min.

9.  Pull off all ethanol with pipet tip.

10. Vacuum dry pellet for 5 min using no heat.

11. Resuspend pellet with 100ul of TE.

12. If multiple reactions, combine them.

13. Store DNA @-80^0^C or -20^0^C.