Quick and Clean Cloning - DNA Cloning and Assembly Methods - page 41

primer 1 primer 2

primer 3 primer 4

lacZ α lacZ α

pUC19 pUC19

BpiI BpiI

BpiI PstI PstI BpiI

A CS

PCR
product

PCR
product

lacZ α

BpiI + ligase

PstI

PstI

A CS

lacZ α

QC cloning vector

PstI

A CS

linearized QC
cloning vector

QC cloning vector

Fig. 2 Preparation of QC cloning vectors. QC cloning vectors can be made by amplifying a lacZ fragment and

a vector backbone fragment from pUC19, and assembling the two fragments using a restriction-ligation with
the restriction enzyme BpiI and T4 DNA ligase. The QC cloning vector is linearized by restriction digest with PstI
before its use for QC cloning

α

be replaced by the user to fi t the adaptor sequence that was
used for amplifi cation of the DNA insert of interest. Since the
same adaptor can be used to amplify different sequences of
interest, primer 1 can be reused for many different cloning
experiments. Primer 2 (ttt gaagac aa gacg ggggaagtagtccttgac
caggcagcccaggg c tgcag tcacagcttgtctgtaagcggatg) has a similar
structure as primer 1 except that the region in italics consists of
a sequence complementary to a fragment of the known region
(sequence K1, Fig. 1b ; in this example, it consists of a frag-
ment of a human immunoglobulin constant region). This
sequence has to be changed by the user to fi t the known
sequence of interest. The size of the catching sequence can
vary from 20 to 50 nucleotides ( see Note 2 ).
2. Amplify the lacZ fragment from pUC19 with primers 1 and 2

using a proof-reading polymerase.
3. Amplify a pUC19 backbone fragment with primers 3 (ttt
gaagac aa cgtc tccgggagctgcatgtg) and 4 (ttt gaagac aa aatg

α