ISOLATE II RNA Micro Kit

Cat No. Size List Price* Qty  
BIO-52074 10 Preps
$62.00
 
BIO-52075 50 Preps
$237.00
 
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ISOLATE II RNA Micro Kit is specially designed for the fast and efficient isolation of extremely pure total RNA from very small samples.

Typical sample material includes tissue (of up to 5 mg) or small numbers of cells (up to 5 x 105) such as laser-captured cells, microdissected cryosections, fine needle aspirates, biopsy samples, cultured cell pellets and flow cytometer sorted cells.

Features & Benefits

  • Rapid protocol - 40 min/12 preps
  • Perfect for small samples - biopsy or single cells
  • High yield and purity - typical A260/A280 ratio 1.9 - 2.1
  • Extra filters (shredders) and DNase I included - for excellent RNA recovery

Applications

Isolation of RNA from small amounts of samples:

  • Single cells
  • Pellets of cultured cells
  • Cryosections
  • Laser-captured or flow sorted cells
  • Small amounts of tissues e.g. biopsy materials

Isolated RNA is ready for downstream applications such as reverse transcription and real-time PCR.


Description

ISOLATE II RNA Micro Kit is specially designed for fast and efficient isolation of extremely pure total RNA from very small samples.

Typical sample material comprises tissue (up to 5 mg), small numbers of cells (up to 5 x 105) such as laser-captured cells, microdissected cryosections, fine needle aspirates, biopsy samples, pellets of cultured cells and flow cytometer sorted cells.

The step-by-step protocol is easy to follow. Cells are lysed with an optimized lysis buffer containing chaotropic ions which simultaneously inactivates RNases, thus preventing degradation of released RNA. The lysate is then applied to a spin column to selectively remove genomic DNA (RNase-free DNase I is supplied with the kit).

There is no need to perform a separate DNase I digestion step. RNA is then bound to the silica membrane. The novel kit design allows elution of RNA in as little as 5–30μl. Simple wash steps remove remaining cell debris. Pure, highly concentrated RNA is eluted in the final step with RNase-free water (fig. 1).

Carrier RNA- poly(-A) RNA is included in the kit, which typically does not interfere with subsequent RT-PCR but allows for optimal performance with the smallest samples.

The isolated RNA shows excellent performance in downstream applications such as reverse transcription, primer extension, Real-Time PCR (fig. 2), Northern blot analysis, microarrays and RNA protection assays.


Components

Reagent

10 Reactions

50 Reactions

ISOLATE II Filters

10

50 

ISOLATE II RNA Micro Columns & Collection Tubes

10

50

Collection Tubes (2ml)

30

150

Collection Tubes (1.5ml)

10

50 

Lysis Buffer RLY

2 x 1.8ml

25ml

Wash Buffer RW1

2 x 1ml 

15ml 

Wash Buffer RW2

2ml

7ml

Membrane Desalting Buffer MEM

1.8ml

10ml

Reaction Buffer for DNase I RDN

0.5ml

3ml

DNase, RNase-free (lyophilized)

1 Vial

1 Vial

 Carrier RNA

300µg

300µg

 Reducing Agent TCEP

14mg

3 x 14mg 

RNase-free Water

5ml

15ml

Product Manual

1

1

Bench Protocol Sheet

1

1


Storage & Stability

All kit components should be stored dry and at room temperature. When stored under the recommended conditions and handled correctly, full activity of kit is retained until the expiry date indicated on the outer box label.


Shipping conditions

Shipped at ambient temperature.

Certificates of Analysis (COAs)

Certificates of Analysis for ISOLATE II RNA Micro Kit are grouped by catalogue number and then listed by batch number. Larger pack sizes are listed first. To locate the COA for your product, first find the catalogue number and then the required batch number.

View all 2 COAs for ISOLATE II RNA Micro Kit →


Frequently asked questions

  • The sequencing protocol I am using says that I have to have my DNA suspended in pure water. Do I have to use the elution buffer supplied with the kit?

      The elution buffer is ideal for applications such as restriction enzyme digestion, sequencing and PCR. It is possible to use DNase free water for elution, but you should expect a slightly lower yield
  • I made my PCR product using RANGER DNA polymerase; it is more than 5kb long. Can I still use the ISOLATE II PCR and Gel Kit to purify the product away from the primers?

      Yes, though this may require some optimisation. Warming the elution buffer to 50 or even 70°C before applying it to the column can help to elute long PCR products, but there is the possibility using this adaptation to the protocol that the primers will be eluted too.
  • I have mutagenized my gene of interest using PCR with relatively long primers (> 30bp). Is the ISOLATE II PCR and Gel Kit suitable for removing excess primers and dNTPs from the reaction?

      The ISOLATE II PCR and Gel Kit can be used under standard conditions to separate most PCR primers away from their product. A primer of only 30bp is efficiently separated, but longer mutagenic primers of around 100bp may be separated from the PCR buffer along with the amplicon.
  • My strain of E. coli contains a low copy plasmid. How can I isolate the plasmid DNA?

      The high quality of the columns used in the ISOLATE II kits, including the miniprep kit, allow larger volumes of culture media to be used as starting material. The exact volume to be used depends on the strain and plasmid used in the experiment, but the miniprep columns will cope with the biomass from 5ml of E. coli grown in LB to an OD600 of around 3. When using larger volumes of culture medium, it is important to ensure that as much spent medium as possible is removed from above the biomass pellet after spinning down the culture. If care is taken, up to 15ml of bacterial culture medium can be used as a starting material, which should give a better yield of low copy plasmid DNA. However, double the volumes of buffers P1, P2 and P3 should be used compared to the standard protocol.
  • My plasmid yield from using the ISOLATE II Plasmid Mini Kit is very low, what could be the cause of this?

      The most frequent cause of low yield is that the biomass pellet is not resuspended properly prior to lysis. Clumps of bacteria mean that the lysis solution cannot access all the cells and these unlysed cells are then spun down later in the process. Low yield can also be caused by incomplete transfer of the cleared lysate to column, especially when the white precipitate made on the addition of buffer P3 is loose, making it hard to retrieve the maximum amount of lysate. Counterintuitively, very high biomass concentrations can lead to low plasmid yield. This can be because the column becomes overloaded, so the yield per mg biomass is less than expected. However use of rich media such as SOC or TY instead of LB can give high biomass but very low plasmid concentration.
  • Another lab has sent me a plasmid, but the purity looks low. Can I use your ISOLATE II Plasmid Mini Kit to clean up the plasmid?

      The ISOLATE II Plasmid Mini Kit can be used to both purify and concentrate naked plasmid DNA. The DNA can be loaded directly onto a fresh column then the remaining washing, drying and elution steps followed as for the standard protocol. The sample can be concentrated by reducing the elution volume in the final stage. However, a better recovery can be obtained by using the ISOLATE II PCR and Gel Kit and treating the plasmid sample as though it were a PCR product.
  • Can I use a gel to quantify RNA isolated using the ISOLATE II RNA kits?

      Agarose gel analysis of RNA samples can be both valuable and misleading. The pattern of bands on the gel can be indicative of the quality of the sample, but equally it can also only indicate that the gel tank, buffer or agarose is contaminated with RNase. A safer measure is to use a spectrophotometer (ideally a microfluidic one) to determine the ratio of A260 to A280 for purity determination. Some microfluidic devices can also give an estimate of the integrity of the RNA. All these methods give an idea of the quality of total RNA which tends to be dominated with rRNA and tRNA. It is not uncommon for an agarose gel to show an abundance of 18S and 23S rRNA but on analysis for there to be little of the transcript of interest. Ideally RNA quality control should include an assessment of the presence of common transcripts (from reference genes, such as GAPDH) using RT-PCR or RT-qPCR.

      If the yield of RNA is low, it is best to first check that all the solutions used and the equipment employed is largely free of RNase. Solutions should be prepared with the highest quality reagents available, preferably ones that have a certificate of analysis to show that they are free of DNase, and RNase. If there is confidence that RNase contamination is at a minimum then it may be worth ensuring that the sample is properly homogenised before lysis, and to check a sample of the lysed material under a light microscope to ensure that all the cells are disrupted.

      RNA samples that have been contaminated with RNase (either during processing or those that have been sent from another lab) can be purified using the ISOLATE II RNA Micro Kit.
  • Can I use columns or solutions from one kit in another one?

      The columns supplied with the ISOLATE II kits are superficially similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.


Customer Testimonials

"My aim is to isolate as much RNA as possible from a small amount of primary cells, which then are used for further qPCR experiments.
With the help of Bioline products (ISOLATE II RNA Micro Kit and SensiFAST cDNA Synthesis Kit) it is possible to achieve the best results with the lowest amount of sample."

Other researchers use:


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