From Sci-Mate Wiki

Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Cloning

---

Editor:  Christopher Dyer Branch: Feedback-Wiki Type: Guidelines Intention: For Publication Read/Write Permissions: Open Access

Authors : 13.09.2010 - Christopher Dyer  -  Sci-Mate

---

Ongoing...

An easy way to insert a gene or sequence of DNA into a Gateway vector is by adding Gateway ends to a PCR fragment, and Gateway cloning the fragment into a Donor vector via the basic Gateway cloning reaction. Once the sequence is within the Gateway vector, it can be very easily moved between vectors for expression, sequencing, tagging, and other sub-cloning purposes via the basic Gateway cloning reaction.

Contents 1 Overview 2 Designing Primers 3 PCR 4 Ligation 5 Transfection

[edit] Overview

ote, however, that Donor Vectors do not express DNA. A second Gateway cloning reaction is needed to move the fragment across to the expression vector. This is not at all difficult, and should not put you off the technique. There are good reasons for this extra step, which are briefly discussed in the general Article.

While this article is limited to getting your fragment into pDONR, designing your cloning experiment should start by considering the final destination of your fragment. Basic design is covered in the general Article, and should be enough for most biologists to get started.

[edit] Designing Primers

Gateway pDONR primers are like any other PCR primers designed for cloning, except they contain a sequence for BP cloning.

The following sequence (B1) is added to the forward primer: 

  5`-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TNN- rest of forward primer.

AND, the following sequence (B2) to the reverse primer:

  5`-GGGG-AC-CAC-TTT-GTA-CAA-GAA-AGC-TGG-GTN- rest of reverse primer.

Some points to consider:  

• Recommended actual PCR primer length is 18-25bps (forward and back).
• The 5`-GGGG end is there to protect the important part of the primier from degradation.
• The two additional nucleotides at the 3` end of the forward primer are only required to keep the product in frame if the final destination vector has an N-terminal tag; and the single N in the reverse primer will keep the gene in frame with a C-terminal tag. The former possibility can be a useful option to keep open, while the latter must be foreplaned.
  Note: do not use AA, AG, or GA for the NN in the forward primer, as this would create a stop codon (the supplier recommends TC in most examples), and avoid stop codons all together in the reverse primer if you specifically intend to insert the gene into a vector with a C-terminal tag.
  
• The inclusion of a Shine-Dalgarno and/or Kozak sequence into the forward primer to assist expression can assist expression in bacterial and/or mammalian cells. The following sequence contains the Gateway B1 and both Shine-Dalgarno and Kozak sequences, which is a handy option to ensure expression (note: the gene must then be kept in frame with the bold Kozak ATG start codon):

  5`-GGGG-ACA-AGT-TTG-TAC-AAA-AAA-GCA-GGC-TTC-GAA-GGA-GAT-AGA-ACC-ATG-G- rest of forward primer

• Long overhangs can be intimidating the first time, but very rarely is there a problem. There is a method, called TOPO cloning, for those afraid of long overhangs, but adding the TOPO is an additional step requring additional TOPO reagent.
• ALWAYS USE A GOOD PROGRAM TO CHECK YOUR ORF IS THE GENE YOU WANT IT TO BE.

[edit] PCR

There is nothing special about the PCR in a Gateway experiment.

Of course, use a proof-reading polymerase. I found the products from Finnzymes to be excellent.

A 50ul reaction is more than enough to run on a gel to check for amplification and correct size, and to use in the Ligation reaction!

[edit] Ligation

	Ligation (ul)	Positive Control (ul)	Negative Control (ul)
DNA Fragment (20-50 fmol)	1-7		1-7
pDONR (150 ng)	1	1	1
pEXP7-tet (as supplied)		2
TE	0-6	5	2-8
BP Clonase II	2	2

BP clonase is expensive, but it works first time almost every time. However, if you calculated the real costs associated with normal cloning (time required, more reagents, repetition, etc), this would be far far higher than this one reagent.

Of course if the original source of the fragment contains the same resistence as your end product, you will want to get rid of it somehow - purification of the DNA fragment from the gel via a kit or silica is an option.

[edit] Transfection

You should expect to see hundreds of colonies on your test plate, and none on your controls.