Restriction digest part II #

After digest on Friday indicated that the plasmid we thought was pMal-hRNASEH1 actually seems to be pcDNA3-hRH1 doing a follow up digest to confirm that.

Protocol #

All units are in micro liters.

Reagent Name	Control	Digest 1	Digest 2
DNA	1.17	1.17	1.17
10x 2.1 buffer	2	2	2
NdeI	0	0.5	0
HindIII	0	0.5	0
EcoRI	0	0	0.5
RstI	0	0	0.5
H2O	16.83	15.83	15.83

Incubate all samples 1 hr at 37 C. Run on 0.8 % gel for 1 hr at 90 v. 1 ul DNA ladder. Samples loaded with 4 ul purple loading dye .

Expected results #

MW:  1 kb DNA Ladder

1:  pcDNA3-hRH1
       1. 6212 bp

2:  pcDNA3-hRH1
    EcoRI + PstI
       1. 4045 bp
       2. 2167 bp

3:  pcDNA3-hRH1
    HindIII + NdeI
       1. 5807 bp
       2. 405 bp

Gel image #

Yep looks like its pcDNA3-hRH1, seeing expected bands at 2 and 4 kb with the EcoRI and Pst digest.