CLONING FROM BEGINNING TO END 详细的克隆操作过程【UCSF】

1. Digest 10 - 20ug DNA to isolate either the desired insert or vector.

2. After digesting, sometimes it is necessary to dephosphorylate the ends of the vector so as to prevent religation before the insert can ligate to vector. If this is necessary:

   VECTOR DEPHOSPHORYLATION (AP/CIP):

   1. after digesting (it is best to do this before running on the gel and Gene Cleaning so no re-ligation can occur during these steps), precipitate the DNA with EtOH (2X the original volume) and NaOAc (10% of original volume).

   2. Place at 4 degrees for 10', then spin at 4 degrees at 13,000 rpm for 20'.

   3. Once you have a pellet, resuspend with dW

   4. Add Alkaline Phosphatase with buffer (see below). 1ug DNA requires a 10 ul reaction (scale up as desired)...

      Reaction should end up: 1 unit Alkaline Phosphatase(AP or "CIP")/ 1ug DNA
      Dilute 10X buffer to 1X
      Bring up to volume with dW
      example: For 20 ug pellet - resuspend in 70 ul dW
      Add 20 ul AP (1unit/ul)
      Add 10 ul 10X buffer (so 1X)
      

3. Add dye (15% Ficoll, 0.25% Bromo Phenol Blue, 0.25% Xylene) at 1:10 to make digests heavy enough to sink into gel wells.

4. Run the digests out on a gel made with "Seakem" brand agarose (it is higher quality than technology grade) at 80-100V, and look at the bands under UV light. Cut out appropriate bands and Gene Clean them (SEE BIO 101 gene clean kit for instructions).

5. If it is necessary to make the ends of the cut insert/vector "blunt ended" for the ligation, now is the time to do it. For this, T4 polymerase is used in combination with the necessary nucleotides (ATGC) to fill in the "sticky ends" with base pairs. The protocol is as follows.

1. For 800ng of a gene cleaned fragments, The reaction is:
   • T4 polymerase
     
   • 10X buffer
     
   • BSA
     
   • GATC
     
   • distilled water to a final volume of 20ul
     
2. Allow reaction to proceed for 20' in a 16 degree water bath.

3. Bring volume up to 100 ul with distilled water and phenol chloroform extract.

   -add equal volume of phenol chloroform, vortex and spin for 5' at 4 degrees. Transfer supernatant to new tube.

4. Add 10ul 3M NaOAc. Vortex.

5. Add 220ul 100% EtOH. Vortex.

6. Place on ice 10' and spin @ 13,000 fro 15' @ 4 degrees.

7. Discard supernatant and resuspend pellet in 10ul TE pH 8.0.

1. Once you have the Gene Cleaned DNA, put together the ligation as follows.

2. Combine the DNA so that you have a ratio of insert to vector at 3:1. The rest of the reaction consists of T4 Ligase, 10X Ligase Buffer, and distilled water. A typical recipe for one reaction is :

   10X Ligase Buffer	1ul
   T4 Ligase	0.5ul
   Vector	1ul
   Insert	3ul
   Distilled water	4.5ul (or bring to 10ul final volume)

3. Mix reactions and put in a 16 degree water bath overnight. It is good to include a reaction mix with vector alone to make sure you have something other than religated vector once you transform and plate them.