UCE: A uracil excision (USER™)-based toolbox for transformation of cereals

Table 1 Comparison of different cloning techniques

	USER™ cloning	Cloning by ligation	GATEWAY^® cloning
Requires PCR cleanup when cloning PCR fragments	No	Yes	No^a
Fragments are allowed to contain restriction enzyme recognition sites	Yes	No^b	Yes
Number of individual PCR fragments that can be cloned simultaneously in one step	> = 3^c	1	1^d
Steps required for cloning into a binary destination vector	1	1 - 2^e	2^f
Time required for ligation/recombination of PCR product(s)	40 minutes	1 - 16 hours^g	1 - 16 hours^h
The technique allows to make constructs without footprints between assembled fragments	Yesⁱ	-	No^d
Length of overhang in primers used for cloning of PCR products	9 bp	5-10 bp^j	> 20 bp

a) Cloning may be done without PCR cleanup, however the manufacturer recommends cleaning PCR products before the recombination reaction
b) Fragments cannot contain recognition sites for the restriction enzymes used in the cloning
c) We have demonstrated the simultaneous cloning of three PCR fragments. More fragments may be possible
d) Simultaneous recombination of multiple entry clones may be used to clone up to four fragments into an expression clone by MultiSite GATEWAY^® cloning. However, this requires two cloning steps and leaves a footprint from the recombination sites between the fragments.
e) Depends on the vector system
f) GATEWAY^® cloning requires that PCR products are cloned into an entry clone before it can be recombined into a destination vector
g) Most protocols recommend ligation for 1-2 hours a room temperature or overnight at lower temperatures
h) Recombination time varies from 1-2 hours to overnight or longer depending on the size of the PCR fragment
i) Multiple fragments cloned in a single step do not have footprints between them. However, sequential USERï¿½ cloning will leave a footprint between fragments.
j) The length depends on what restriction enzymes are used

ISSN: 1746-4811