西亚试剂：：.Spin Column Chromatagraphy

Remove enzymes by spin chromatography using QIAquick PCR Purification Kit #28106
pg. 18 of the QIAquick Spin Handbook.

V.Ligation of BSTX1 Adapters

Note:Thaw adaptors on ice--be careful to never warm adapters.
Speed vac your 30µl DNA sample down to 23µl. To setup the ligation of BAC insert to adapters, add the following components to a 1.5ml microfuge tube.
Set up with all tubes on ice:

Incubate overnight at 16°C.

VI.Spin Column Chromatagraphy #1

Heat inactive adapter ligation reaction at 65°C for 5 minutes.

Remove excess adapters and enzymes by spin chromatography using QIAquick PCR Purification Kit #28106 pg. 18 of the QIAquick Spin Handbook.
Elute 1X in 30µl preheated EB, 1X in 30µl preheated water.

VII.Gel Purification #2

To ensure that all adapters are removed from BAC insert, do a final gel purification on the column-purified DNA. I have tried to elimated this step, but found that not all of the adapters were removed if I just did a column purification.

Run DNA on a 0.7% Agarose gel (use High quality agarose and TAE buffer). Before cutting out bands, rinse gel with dH2O to get rid of any adaptors that may be in gel buffer.

Gerl purify using the protocol from Qiagen's QIAquick Spin Handbook entitled "QIA gel Extraction Kit Protocol. Include Step #9 and elute in 1X 30µl EB, and 1X in 30µl dH2O. Note, try to run gel such that DNA is contained within 2-3 gel slices. If DNA extracted from too many gel slices and eluants are combined and concentrated, we ahve noted that ligation efficiency goes down.

VIII.Ligation to pIK 96 vector

Note, pIK96 DNA should be prepared according to the Stanford protocol. The amount of vector DNA used in the ligations depends on the concentrations of that particular batch of vector, so adjust accordingly. ( I like to use approximately 100-200ng vector DNA).

Incubate overnight at 16°C (can even go all weekend, if need be).

IX.Transformation into Stbl2 Chemically Competent Cells

Heat inactivate ligation at 65°C for 5 minutes. Check 42°C incubator to make sure it is at the correct temperature. Keep ligation on ice until ready to transform.

Using Invitrogen MAX Efficiency StBl2 Competent Cells, use the following transformation procedure:

1. Thaw competent cells on ice. Place the required number of Falcon 2059 tubes on ice for each transformation that you are doing.

*Falcon 2059 tubes or other similarly shaped 17 X 100 mm polypropylene tubes are required for optimal transformation efficiency.

2. Dilute each ligation 1:5 in 10mM Tris-Cl, pH 7.5 and 1mM EDTA.

3. Add 100µl of the Stbl2 cells to the Falcon 2059 tube. NOTE: Handle cells very carefully and do not allow them to warm! Add 1.0µl of the diluted ligation to the Falcon 2059 tube that contains the competent cells.

4. Incubate the Falcon 2059 tubes containing the cells and DNA on ice for 30 minutes.

5. Heat shock at 42°C for 25 seconds.

6. Incubate on ice for 2.0 minutes.

7. Carefully add 900µl of S.O.C. media to the cells.

8. Shake tubes at 225 rpm, at 30°C for 90 minutes.

9. Plate desired quantity of culture on an LB Carb 100 plate with 5% sucrose. With new transformations, try plating several amounts of cells (ex. 50µl, 150µl).

Plate pik96 + SacB control on LB-Carb/Sucrose and LB Carb.
Plate pik96 control on LB/Carb/Sucrose plate.

10. Allow colonies to grow overnight at 30°C or 24 hours at 28°C.

*Note: The Stbl2 cells seem to be sensitive to time left on plate, and so it is best to pick colonies off fresh plates. If colonies are used that are even a few days old, they will not grow as well

丙烯醛肉桂醛桂皮醛4-硝基肉桂醛