Validating GST entry clones

The AGRIKOLA consortium has cloned most of the CATMA GSTs into pDONR207, a Gateway entry vector. These entry clones are in E. coli DH5α. pDONR207 confers resistance to gentamycin.

The Gateway cloning within the AGRIKOLA project employed high-throughput protocols that avoided isolation of single colonies during the cloning procedure. Therefore, before going any further you should streak the bacteria out on agar plates containing 15 mg.l^-1 gentamycin and select 4 or 6 individual colonies for verification.

Knowing the size of your chosen GST, you can verify the plasmid by restriction digest using the pDONR207 restriction map or better still by PCR amplification and/or sequencing. We use the primers shown below for validating entry clones. The sequencing of GST inserts in pDONR207 may be tricky, especially for the smaller GSTs. We use an alternative sequencing protocol including a blocking primer for GST sequence validation.

Primer design

                    SeqL-A : 5' TCG CGT TAA CGC TAG CAT GGA TCT C 3'

                    SeqL-B : 5' GTA ACA TCA GAG ATT TTG AGA CAC 3'

These primers can be used for PCR validation and also for sequencing.

PCR on bacterial colonies with primers SeqL-A/SeqL-B

SeqL-A 10 µM	0.2 µl
SeqL-B 10 µM	0.2 µl
10 x Red Cresol (Aldrich cat no. 11447-2)	2 µl
10x PCR buffer	2 µl
dNTP stock (2.5 mM of each dNTP)	0.8 µl
Taq enzyme (home made...)	0.2 µl
water	14.6 µl

PCR conditions

	94°C	5 min
	94°C	30 sec
35 X	52°C	30 sec
	72°C	1 min
	72°C	5 min