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Shotgun Library

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This protocol is intended to make shotgun libraries from BACS that are to be fully sequenced and assembled. To minimize chimeric clones, an adaptor method is used. There are simpler protocols if just random shotgun sequence is desired.

I. Shearing BAC DNA To shear the large insert insert BAC clone, use the Hydroshear by Genemachines. The settings will vary depending on which orifice you are using, and what size fragments you are trying to generate. New orifices need to be "calibrated" to determine proper settings. The standard we use, to generate predominately 2-5kb fragments is: Cycles: 20 Speed code:10-12.

To make sure BAC DNA is in solution, incubate DNA at 37℃ for 30 minutes, vortexing hard every 10 min. Centrifuge at 12,000 rpm for 4 minutes to determine if the DNA is completely in solution. The DNA is not in solution if a clear pellet can be observed. Transfer the top 190µl to a clean 1.5ml microfuge tube, and proceed with shearing. *Note: We have found that the following works well, when BACDNA is isolated from 500mls LB culture, using the Qiagen-500 tips, and resuspending in 600µl T10E1 at the end. Depending on how concentrated the DNA looks on a gel, dilute an aliquot in water. 100µl of BAC DNA diluted in 100µl water has worked well in the past.

Shearing volume should not exceed 200µl

Wash shearing orifices before and after each sample with the following settings:

*All solutions must be filterd with a 0.2 micron filter before using.

0.2 M HCl x2

0.2 M NaOH x3

ddH2O x5

Please use the Hydroshear manual pgs. 45-46 for instructions to shear DNA.

**Important: Before shearing, DNA must be in solution, or the Hydroshear will clog.

II. DNA Precipitation

After shearing, precipitate DNA with 1/10 volume 3M NaOAc, and 2 volumes ethanol. Spin and wash with 70% ethanol. Dry pellet and resuspend in 150µl water.

III.Repairing ends of the sheared DNA

Add the following components to a microfuge tube:

Sheared DNA (10-50g)
dH2O
dNTPs (25mM mixture of 4 dNTPs)
BSA (10mg/ml)
10X T4 DNA Polymerase Buffer
T4 DNA Polymerase
Total Vol.
150.0µl
23.5µl
1.5µl, final conc. = 125µM
1.0µl, final conc. = 50mg/m
20.0µl
4.0µl, 12 units
200µl
Incubate at 16℃ for 1 hour.

Incubate at 65℃ for 5 min. to inactivate the enzyme.

Precipitate the BAC DNA with 1/10 volume 3M NaOAc pH 5.2 and 2 volumes 100% EtOH.

Sit on ice for 15 minutes

Centrifuge for 30 minutes at 14,000rpm.

Carefully wash pellet with 1.0ml 70% EtOH. Centrifuge 5 min.

Dry time for pellet is usually 5-10 minutes.

*Note, it's important to not let the pellet over dry, or it will be difficult to get into solution!

Resuspend BAC DNA in 61.5µl of sterile, autoclaved water. Pipette up and down to ensure the pellet is in solution. Heat at 42 for 10-15 minutes.

To phosphorylate any 5'-OH ends, add the following components to a 1.5ml microfuge tube: Thaw ATP on ice (make a bunch of aliquots rather than keeping rethawing)

 

T4 DNAP-repaired BACDNA
10X PNK buffer
25 mM ATP
T4 PNK kinase (10,000 units/ml)
61.5µl (~30µg)
7.5µl
3.0µl final concentration = 1mM
3.0µl (20 units)
Incubate at 37℃ for 30 minutes.

Incubate at 65℃ for 5 minutes to inactivate the enzyme.

IV.Spin Column Chromatagraphy #1

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:

DNA
10X T4 DNA Ligase buffer
BstXI adapter (0.5µg/µl)
T4 DNA ligase (400,000µg/ml)
Total
23.0µl
3.0µl
2.0µl
2.0µl
50.0µl
Incubate overnight at 16℃.

VI.Spin Column Chromatagraphy #1

Heat inactive adapter ligation reaction at 65℃ 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).

Insert DNA

Insert DNA
pIK96 vector
T4 DNA 10X ligation buffer
T4 DNA Ligase (400,000 µ/ml)
Total
15µl
2.0µl
2.0µl
1.0µl
20.0µl
Incubate overnight at 16℃ (can even go all weekend, if need be).

IX.Transformation into Stbl2 Chemically Competent Cells

Heat inactivate ligation at 65℃ for 5 minutes. Check 42℃ 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℃ 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℃ 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℃ or 24 hours at 28℃.

*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.

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