Subcloning inserts from traditional BAC or PAC vectors into

Thursday, December 04, 2003

Description
A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid,and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. Published examples include a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment inserted into the BIBAC vector and transformed to plants.

Procedure
Do a CsCl prep of the BIBAC vector
Do a CsCl prep of the BAC clone of interest.

Cut the BAC clone with NotI.
Cut the BIBAC with NotI.

Quantify the BIBAC NotI DNA compared to the high molecular weight BAC NotI fragment. We use lambda DNA as a standard loaded at 20, 50, 100, and 200 ng on a standard gel.

Treat BIBAC NotI with SAP (shrimp alkaline phosphatase USB) or other phosphatase (CIAP BRL).

Set up a series of ligations using BIBAC vector with and without phosphatase and with a ration of vector: insert of about 10 : 1 and 5 : 1.
I recommend that you set up a couple of different ratios because estimating the concentrations is not always accurate.

Here is an example of a (successful) ligation mix. BIBAC2 vector NotI + CIAP 200 ng 4 ul
BAC clone NotI (pBeloBAC or derivative)
= vector (CmR 7 Kb) + insert (128 Kb) 100 ng 7 ul
10X ligation buffer (NEB) 10 ul
T4 DNA ligase (NEB) 2 ul
high quality water
(distilled, deionized) to make 100 ul 75 ul

Mix DNA + vector + water (gently) incubate at 60 C for 10 minutes then remove to room temp.
Mix in 10X ligation mix + enzyme incubate overnight at 16 C.

Drop dialyze a portion of the ligation mix (30 ul) against water at room temp for 30 minutes (not essential, but you will get more colonies from your electroporation).

Use 1 ul for electroporation of DH10B cells.

When subcloning, I usually plate out on Kan and then replica plate to test for CmR (internal control for ligation) and Kan + sucrose and then print a Kan plate last.

You should get a few colonies that are CmR - they contain the BAC vector - but also serve to tell you that the ligation worked.

From this ligation mix I checked 38 colonies (from one electroporation); 10 were CmR, all were Kan + sucroseR. Apparently some CmR colonies that are sucrose sensitive have also been observed. It is possible that this due to read through of the sacB gene when CmR gene is inserted upstream and in the same orientation - but this has not been verified.

You can also plate a second electroporation onto Kan + sucrose, but you should replica plate these as well. In some batches of competent cells we have observed contaminants that grow on Kan + sucrose and NOT on Kan. Also you can always manage to generate "garbage"s (deletions, rearrangements) in any "bad" ligation mix.

Analyze CmS, Kan + SucR colonies by standard BAC mini-prep. For this experiment 16 were checked on a pulsed field gel. Eight of these that appeared to have an insert of the correct size were then cut with EcoRI and run on a standard gel to compare the restriction pattern with that of the original BAC clone.

Modifying the BIBAC vector

(eg, introduction of a desired plant selectable marker into pCH20, or BIBAC1)

Do a 2 liter prep of the (BIBAC) plasmid to be modified.

Restriction enzyme digestion is by standard methods.

Isolation (if needed) of fragment to be inserted into the BIBAC is by standard methods (eg prep-a-gene, gelase).

We use Klenow to "chew back" 3' overhangs as well as to fill in 5' overhangs to blunt ends as needed. We use standard reaction conditions for both activities.

Ligations are standard but are likely to be blunt-end ligations. For blunt end ligation s I recommend that PEG (10%) be included in the ligation mix or use a commercial T4 DNA ligase buffer that already has PEG (eg BRL) which serves as to exclude volume, increasing the concentration of ends in the reaction mix.

I usually set up a series of ligations
I use 20-50 nanograms of vector in a 10 microliter (final volume) ligation mix. Fragment is added in a ratio of 5-10 molar excess of fragment (insert) to vector.

You can set these up with and/or without treating with phosphatase. In my hands CIAP is more "perfectly" efficient (and we use it for library construction), but for standard cloning I prefer SAP for the ease of heat inactivation and better activity on blunt ends.

[I usually set the ligations up both + and - SAP.]

You should include standard controls, in particular, vector no ligase. Incompletely digested vector is a common source of background. In cloning experiments that have no selection for the desired product, this becomes significant.

When you are not using sacB as a selectable marker (or even if you are, see BAC subcloning section) - I recommend plating transformants on Kan plates and then replica plating to Kan + sucrose. This provides more accurate information (as there is always some background with any marker, and we have observed contaminants in competent cell preps that grow on Kan + sucrose that are not really Kan resistant). If cells with the BIBAC vector are plated on Kan + sucrose, you will get a few colonies that are likely to contain a mutation that prevents expression of sacB. This will occur at a low frequencies under strong selection pressure (survival).

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Subcloning inserts from traditional BAC or PAC vectors into