The GetClone™ PCR Cloning Vector II is a positive selection system for high efficiency cloning of blunt end DNA or amplicons. This cloning vector contains a lethal gene which can be disrupted by ligation of a blunt end DNA insert into the cloning site. Only colonies with inserted vectors are able to propagate, eliminating the additional needs of IPTG and X-Gal for blue/white screening. This cloning vector includes ampicillin and kanamycin resistance genes that can meet the needs of most users.
Detail
Description
The GetClone™ PCR Cloning Vector II is a positive selection system for high efficiency cloning of blunt end DNA or amplicons. This cloning vector contains a lethal gene which can be disrupted by ligation of a blunt end DNA insert into the cloning site. Only colonies with inserted vectors are able to propagate, eliminating the additional needs of IPTG and X-Gal for blue/white screening. This cloning vector includes ampicillin and kanamycin resistance genes that can meet the needs of most users.
Features
Cloning efficiency greater than 90%
IPTG and X-Gal are not required
Accepts a wide range of insert/vector ratios 0.5:1 to 12:1
Accepts insert size from 6 bp to 11 kb
The phosphorylation of PCR fragments is not required
Accepts blunt end amplicon or DNA fragment (not for sticky ends)
Thymidylate Synthase (TS) is a crucial enzyme responsible for the synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP) a precursor for thymidylate which is necessary for DNA replication and repair from 2′-deoxyuridine-5′-monophosphate (dUMP). In terms of cancer, TS is an important target for cancer treatment as the inhibition of TS and therefore nucleotide synthesis necessary for cell growth has shown to be a vital part for successful treatment against colorectal, pancreatic and breast cancers.
AAV Purification from any input – cell fraction or media fraction
High AAV recovery, up to 90%
No specialized equipment needed
Purification from a variety of AAV serotypes (including AAV6 and AAV9)
Yields highly active AAV for in vivo and in vitro experiments
Purification is based on spin column chromatography that uses Norgen’s resin separation matrix
Recombinant adeno-associated virus (AAV) vectors are highly promising tools for both in vitro and in vivo gene transfer. Norgen’s AAV Purification Kits provide fast and simple procedures for concentrating and purifying AAV vectors from cell lysate and cell culture media. Purification is based on precipitation onto Norgen Biotek’s proprietary resin. Contaminating cellular debris is largely removed from the sample via a centrifugation step, while contaminating DNA and RNA is reduced using enzymatic digestion. AAV vector purified in this manner is highly active for use in in vitro and in vivo transduction experiments.
AAV Purification Kit
Norgen’s AAV Purification Kit contains sufficient materials for 15 preparations (33.5 mL per prep of supernatant (SN) or a total of 500 mL of supernatant input). Approximately 1 mL of cell pellet can be purified per prep, up to a maximum of 15 mL of cell pellet in total for the entire kit. Up to 33X sample concentration.
AAV Purification Mini Kit
Each spin column is able to concentrate and purify AAV from 0.5-8 mL of cell pellet, cell culture media, or cells and culture media mixed together. Up to 50X sample concentration. AAV vector purified in this manner is highly active for use in in vitro transduction experiments, and is eluted into a small volume (200 µL). Preparation time for 4 samples is 1.5 hours, with 45 minutes of hands-on time.
AAV Purification Midi Kit
Each spin column is able to concentrate and purify AAV from 8 mL up to 45 mL of input consisting of cell pellet, cell culture media, or cells and culture media mixed together. Up to 50X sample concentration. AAV vector purified in this manner is highly active for use in in vitro transduction experiments, and is eluted into a small volume (1 mL). The kit may be used to purify up to 8 x 25 mL or 4 x 45 mL of samples using the included columns. Preparation time for 4 samples is approximately 2 to 2.5 hours, with 1.5 hours of hands on time.
AAV Purification Maxi Kit (Slurry Format)
Each spin column is able to concentrate and purify AAV from 45 mL to 90 mL of input consisting of cell pellet, cell culture media, or cells and culture media mixed together. Up to 200X sample concentration. AAV vector purified in this manner is highly active for use in in vitro transduction experiments, and is eluted into a small volume (1-10 mL) using the optional concentration step. The kit may be used to purify up to 1 x 900 mL samples or 10 x 45-90 mL samples using the included columns. Preparation time for 1 x 900 mL sample is approximately 2.5 to 3.5 hours, with an optional concentration step requiring an additional 30 min.
At least 5 x 1010 AAV particles as determined by qPCR
AAV Vector Serotype
AAV6, AAV9 and others
Input Type
Cells, media
Input Volume (AAV supernatant)
1 – 33.5 mL SN per prep (500 mL SN in total)
Input Volume (AAV cell pellet)
1 mL cell pellet per prep (15 mL in total)
Time to Complete Purifications
2.5 to 4.5 hours with 1 hour hands on time
In vivo transduction
Yes
Storage Conditions and Product Stability
HL-SAN Nuclease should be stored at -20°C upon arrival. Elution Buffer O should be stored tightly capped at 4°C upon arrival. All other solutions should be kept tightly sealed and stored at room temperature. Once opened, the solutions should be stored at 4°C. This kit is stable for 2 years after the date of shipment.
Component
Cat. 66100 (15 preps)
Cat. 63200 (20 preps)
Cat. 63300 (4-8 preps)
Cat. 63250 (1-10 preps)
Lysis Buffer S
5.5 mL
5.5 mL
5.5 mL
20 mL
DNAse I
–
2 x 25 uL
2 x 25 uL
210 μL
RNAse A
–
60 μL
60 μL
240 μL
HL-SAN Nuclease
102 μL
–
–
–
Binding Buffer A
20 mL
4 mL
4 mL
2 x 8 mL
Purification Solution C
60 mL
–
–
–
Purification Solution D
130 mL
–
–
–
Wash Solution C
2 x 130 mL
60 mL
60 mL
3 x 60 mL
Slurry E
12.5 mL
–
–
2 x 14.5 mL
Elution Buffer O
66 mL
8.5 mL
8.5 mL
66 mL
Protein Neutralizer
4 mL
4 mL
4 mL
4 mL
Spin Columns
–
20
–
–
Mini Spin Columns
–
20
–
–
Midi Spin Columns (grey contents) with Collection Tubes
–
–
8
10
Midi Spin Columns (white contents) with Collection Tubes
–
–
8
–
Maxi Spin Columns (grey contents) with Collection Tubes
–
–
–
10
Maxi Spin Columns (white contents) with Collection Tubes
