Download BioModule BLOCK-iT Units with Pol II miR RNAi Expression System

Kit Contents and Storage, Continued
Unit Definition of
T4 DNA Ligase
One (Weiss) unit of T4 DNA Ligase catalyzes the exchange of 1 nmol 32P-labeled
pyrophosphate into [γ/β-32P]ATP in 20 minutes at 37°C (Weiss et al., 1968). One
unit is equal to approximately 300 cohesive-end ligation units.
Primer Sequences
The table below provides the sequence and the amount of primers included in
the kit.
Primer
EmGFP forward
sequencing primer
miRNA reverse
sequencing primer
Sequence
5′-GGCATGGACGAGCTGTACAA-3′
Amount
2 µg
(323 pmol)
5′-CTCTAGATCAACCACTTTGT-3′
2 µg
(332 pmol)
LacZ Control Oligo The sequences of the miR-lacZ positive ds control oligo are listed below. The miRlacZ top and bottom strand oligos are annealed and are supplied in the kit as a
Sequences
10 nM double-stranded oligo that is ready-to-use in the ligation reaction (page
51).
LacZ DNA Oligo
Sequence
Top strand
5’-TGCTGAAATCGCTGATTTGTGTAGTCGTTTTGGCCACTGACTGACGACTACACATCAGCGATTT-3’
Bottom strand
5’-CCTGAAATCGCTGATGTGTAGTCGTCAGTCAGTGGCCAAAACGACTACACAAATCAGCGATTTC-3’
One Shot® TOP10
Reagents
The following reagents are included in the One Shot® TOP10 Chemically
Competent E. coli kit (Box 2). Transformation efficiency is ≥ 1 x 109 cfu/µg
plasmid DNA. Store Box 2 at -80°C.
Reagent
Composition
S.O.C. Medium
2% Tryptone
(may be stored at +4°C or
room temperature)
0.5% Yeast Extract
Amount
6 ml
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM glucose
Genotype of
TOP10 Cells
21 x 50 µl
TOP10 cells
--
pUC19 Control DNA
10 pg/µl in 5 mM Tris-HCl, 0.5 mM 50 µl
EDTA, pH 8
F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu)7697
galU galK rpsL (StrR) endA1 nupG
Continued on next page
ix