Biomedical Engineering Reference
In-Depth Information
A1 Tag:
5
GAA TTC ACA AGT TTG TAC AAA AAA GCA GGC TGG
ATG XXX XXX XXX3
A2 Tag:
5
GTC GAC CAC TTT GTA CAA GAA AGC TGG GTG
CTA XXX XXX XXX 3
Sequences in bold represent insert specific sequences (X). Other stop codons can be used
as required; 18-21 gene-specific nucleotides must be added in-frame with forward and reverse
tag sequences. When generating PCR inserts from clones in the pDONR223 vector the following
primer pairs should be used:
Forward primer:
5
GAATTCACAAGTTTGTACAAAAAAGCTGGCATG 3
Reverse primer:
5
GTCGACCACTTTGTACAAGAAAGCTGGG 3
b
DNA concentration can be estimated by measuring absorbance at A
260
on a UV spectropho-
tometer or by comparison with the intensity of band in molecular weight markers.
c
If a single band of the correct size is obtained, the PCR product can be used directly in gap
repair reactions, without further purification. However, if multiple bands are produced, or if
bands are very weak, it is advisable to purify and concentrate the required band. Alternatively,
PCR reactions containing multiple bands can be used directly in gap repair reactions, as yeast
containing vectors with the correct sized insert can be identified when performing subsequent
yeast colony (YC) PCR reactions (Sections 8.2.4 and 8.2.5 below). Annealing temperatures and
extension times may need to be optimized for each template in accordance with instructions
provided with the hot-start KOD polymerase.
8.2.3 Performing gap repair reactions
Gap repair reactions require host cells to be transfected with a combination of linearized
vector and complementary recombination-compatible PCR products (see Protocol 8.2).
PROTOCOL 8.2 Gap Repair Reactions
Equipment and reagents
•
PCR tubes
•
PCR machine
•
Appropriate selective (low ade) plates
•
1
M
LiOAc
•
QIAquick PCR purification kit (250)
•
Filter-sterilized 50% (w/v) PEG 3350
•
Sterile water
Salmon testis carrier DNA (heat denatured at 99
◦
C for 5min before use)
d
•
