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Inducible shRNA Lentiviral Vectors

互联网2013-11-13

1283

实验原理

A large selection of BLOCK-iT™ RNAi products is available from Invitrogen to facilitate RNAi analysis in mammalian and invertebrate systems. The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit supplied with the BLOCK-iT™ Inducible H1 Lentiviral RNAi System uses a vector-based approach to allow efficient generation of H1/TO RNAi cassettes for regulated expression of shRNA molecules in mammalian cells. For constitutive expression of shRNA molecules in mammalian cells, use the BLOCK-iT™ U6 RNAi Entry Vector Kit or the BLOCK-iT™ Lentiviral RNAi Expression System. Other BLOCK-iT™ RNAi products are available to facilitate production and delivery of synthetic short interfering RNA (siRNA), diced siRNA (d-siRNA) or double-stranded RNA (dsRNA) for RNAi analysis in mammalian cells or invertebrate organisms, as appropriate. For more information about any of the BLOCK-iT™ RNAi products and other RNAi resources, see the RNAi Central application portal at www.invitrogen.com/rnai

实验步骤

1. Performing the LR Recombination Reaction

1) Add the following components to 0.5 ml microcentrifuge tubes at room temperature and mix.

Component	Sample	Positive Control
Entry clone (50-150 ng/reaction)	1-7 µl	--
pENTR^™ -gus (50 ng/µl)	--	2 µl
pLenti4/BLOCK-iT^™ -DEST vector (150 ng/µl)	1 µl	1 µl
TE Buffer, pH 8.0	to 8 µl	5 µl

2) Remove the LR Clonase™ II enzyme mix from -20° C and thaw on ice (~ 2 minutes).

3) Vortex the LR Clonase™ II enzyme mix briefly twice (2 seconds each time).

4) To the sample above, add 2 µl of LR Clonase™ II enzyme mix. Mix well by pipetting up and down.

Reminder: Return LR Clonase™ II enzyme mix to -20° C immediately after use.

5) Incubate the reaction at 25° C for 1 hour.

Note: Extending the incubation time to 18 hours typically yields more colonies.

6) Add 1 µl of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37° C.

7) Proceed to Transforming One Shot® Stbl3™ Competent E. coli

Note: You may store the LR reaction at -20° C for up to 1 week before transformation, if desired.

2. Transforming One Shot® Stbl3™ Competent E. coli

1) Thaw, on ice, one vial of One Shot® Stbl3™ chemically competent cells for each transformation.

2) Add 2 to 3 µl of the LR recombination reaction into a vial of One Shot® Stbl3™ cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1 µl) of DNA into a separate vial of One Shot® cells and mix gently.

3) Incubate the vial(s) on ice for 30 minutes.

4) Heat-shock the cells for 45 seconds at 42 °C without shaking.

5) Remove the vial(s) from the 42 °C water bath and place them on ice for 2 minutes.

6) Add 250 µl of pre-warmed S.O.C. Medium to each vial.

7) Cap the vial(s) tightly and shake horizontally at 37 °C for 1 hour at 225 rpm in a shaking incubator.

8) Spread 25-100 µl of the transformation mix on a pre-warmed selective plate and incubate overnight at 37 °C. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium and plate 25-100 µl.

9) Store the remaining transformation mix at 4 °C. Plate out additional cells the next day, if desired.

3. Producing Lentivirus in 293FT Cells

1) Grow the 293FT Cells to obtain 6 x 10⁶ 293FT cells for each sample.

2) Prepare plasmid DNA of your expression clone.

3) Cotransfect the ViraPower™ Packaging Mix and pLenti6/V5-GW/miR expression plasmid DNA into 293FT Cells using Lipofectamine™ 2000.

4) Harvest virus-containing supernatants 48-72 hours post-transfection.

4. Titering Your Lentiviral Stock

1) The day before transduction (Day 1), trypsinize and count the cells, plating them in a 6-well plate such that they will be 30-50% confluent at the time of transduction. Incubate cells at 37°C overnight.

Example: When using HT1080 cells, we usually plate 2 x 10⁵ cells/well in a 6-well plate.

2) On the day of transduction (Day 2), thaw your lentiviral stock and prepare 10-fold serial dilutions ranging from 10-2 to 10-6. For each dilution, dilute the lentiviral construct into complete culture medium to a final volume of 1 ml. DO NOT vortex.

Note: You may prepare a wider range of serial dilutions (10-2 to 10-8), if desired.

3) Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells (total volume = 1 ml).

4) Add Polybrene® (if desired) to each well to a final concentration of 6 µg/ml. Swirl the plate gently to mix. Incubate at 37°C overnight.

5) The following day (Day 3), remove the media containing virus and replace with 2 ml of complete culture medium.

6) The following day (Day 4), proceed to Steps 7-8 for EmGFP titering method or proceed to Steps 9-14 for Blasticidin titering method.

7) Determine the titer by flow cytometry on Day 4 for titering EmGFP. For each viral dilution well of the 6 well plate, trypsinize and resuspend the cells in complete media at a concentration of 10-500 cells/ml.

8) Using a flow cytometry system, determine the percentage of GFP-positive cells for each dilution.

9) For Blasticidin selection, remove the medium on Day 4 and replace with complete culture medium containing the appropriate amount of Blasticidin to select for stably transduced cells.

10) Replace medium with fresh medium containing Blasticidin every 3-4 days.

11) After 10-12 days of selection (day 14-16), you should see no live cells in the mock well and discrete Blasticidin-resistant colonies in one or more of the dilution wells. Remove the medium and wash the cells twice with PBS.

12) Add crystal violet solution (1 ml for 6-well dish; 5 ml for 10 cm plate) and incubate for 10 minutes at room temperature.

13) Remove the crystal violet stain and wash the cells with PBS. Repeat wash.

14) Count the blue-stained colonies and determine your lentiviral stock titer.

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