多基因克隆方法（多DNA片段组装）

To construct the final plasmid from the six starting materials (gene 1–4, replication origin and a selectable marker), SFs are first prepared by linking every two materials together, usually suing overlap-extension PCR (OE-PCR). So as shown in this figure, every SF has its 3′-half overlapped with the 5′-half of the next SF and the 5′-half of the first SF overlaps with the 3′-half of the last SF. A mixture of these SFs was denatured at 100°C to free all single strands. When it cools back down to room temperature, annealing between the overlaps would assemble the single strands one after another into a cycle which can be further repaired into double-stranded, closed circular molecule after transformation into the cells.
http://www.plosone.org/article/i ... ournal.pone.0030267

PCR-after-ligation method for cloning of multiple DNA inserts

PCR-after-ligation method for cloning of multiple DNA inserts
From  Sciencedirect
http://www.sciencedirect.com/sci ... i/S0003269710002198

Outline of the PCR-after-ligation method for efficient multiple DNA insert cloning. The DNA inserts and vector are digested with restriction enzymes to obtain compatible termini, followed by purification and ligation (step 1). Most of the products obtained should be either non-full-length DNA fragments or inverted repeat fragments by self-ligation. Then PCR is performed to amplify ligationproduct using flanking primers, and the DNA fragment with expected size is obtained by gel purification (step 2). The purified DNA fragment is inserted into the linearized vector, followed by transformation into E. coli (step 3).

Agarose gel shows the products obtained from PCR-after-ligation. Lane M: DNA size marker; lanes 1–3: PCRproducts amplified by using the ligationproducts with different molar ratios of vector/inserts as templates. The molar ratios of vector/inserts for lanes 1, 2, and 3 are 1:1:1:1:1, 1:2:3:4:5, and 1:3:1:3:1, respectively.

Gel extraction of DNA fragments running close together on your agarosegel

Gel extraction of DNA fragments running close together on your agarosegel
from fermentas

If the DNA fragment you would like to extract from a Gel is covert or run very close to a second DNA fragment you can perform a restriction digest with your DNA fragments before loading them on the gel. With the FastDigest® Restriction enzymes in a 5 min reaction.
Choose a restriction enzyme which only cuts in the DNA fragment you are not interested in. The fragment will now be much smaller and will not migrate together with your DNA fragment of interest any more. It is much easier to extract.
You can use the REviewer™ tool on the Fermentas homepage to paste in both sequences and analyse easy what FastDigest® enzyme to choose.

Closely running fragments can not be extracted without contamination

Digestion of second fragment leads to clear separation of fragment of interest

General recommendations to avoid RNase contamination

From fermentas

    Maintain a separate area, dedicated pipettors and reagents when working with RNA.
    Wear gloves when handling RNA and reagents to avoid contact with skin, which is a source of RNases. Change gloves frequently.
    Use sterile, RNase-free plastic tubes.
    Treat water and all solutions used for RNA purification and handling with DEPC. Add DEPC to 0.1% (v/v) final concentration; incubate overnight at room temperature and autoclave.
    High quality reagents must be used for buffer solutions. Buffers containing Tris should be prepared by dissolving Tris base in DEPC-treated water. Solutions containing DTT or nucleotides should be prepared using DEPC-treated water and be passed through a 0.2 µm filter for sterilization.
    Keep all kit components sealed when not in use and all tubes tightly closed during the transcription reaction.

PCR product clean-up prior to sequencing

PCR product clean-up prior to sequencing
From fermentas

The clean-up reaction removes unincorporated primers and degrades unincorporated nucleotides. The resulting PCR product is ready to use for sequencing without additional purification, e.g., using column purification kits.


    Prepare the following reaction mixture:
    PCR mixture (directly after completion of PCR)         5 µl
    Exonuclease I (#EN0581)         0.5 µl (10 u)
    FastAP™ Thermosensitive Alkaline Phosphatase (#EF0651) or
    Shrimp Alkaline Phosphatase (#EF0511)         1 µl (1 u)

    Mix well and incubate at 37°C for 15 min.
    Stop the reaction by heating the mixture at 85°C for 15 min.

Note

    Up to 5 µl of purified PCR products can be used directly for DNA sequencing without further purification.
    For reliable sequencing results there should not be non-specific PCR products.
    The protocol may be applied for clean-up of PCR products, generated by any thermophilic DNA polymerase or polymerase mix.
    The procedure is not recommended for downstream cloning applications.

Determination of RNA fragment lenghts in Northern Blots without labelled markers

Determination of RNA fragment lenghts in Northern Blots without labelled markers
From Fermentas

1.) Load the RNA marker on the Northern Gel beside your samples

Important: Use the same loading dye for the marker and your samples AND load same volumes. Adjust the volumes with DEPC water

2.) Run the gel and blot the RNA. Crosslink the RNA on the membrane
3.) Cut off the marker lane

Possibility A: Staining with methylene blue

4.) Put the membrane containing the marker lane into a clean, flat bowl. Add methylene blue solution. The membrane must be completely covered (ideally, the gauge should be around 0.8 cm)

5.) Shake the bowl very carefully for about 5-10 min

6.) When the RNA marker bands appear blue, discard the methylene blue solution and wash the membrane with tap water (optionally, 2 x 30 s). The methylene blue solution can be reused several times (store at 4°C)
7.) The marker lane can be a) scanned beside the developed blot or b) mark with a pen the marker bands on the blot (pen writings are mostly visible on fluorescence scanners)
Methylene blue solution: e.g. the Methylene blue solution from MRC (Molecular Research Center), distributed by Fermentas

Possibility B: Visualizing with UV light

4.) Put the membrane containing the marker lane on an UV screen. Mark with a pen the marker bands and the marker lane can be scanned beside the developed blot

Analysis of ligation products by agarose gel electrophoresis

fermentas
Analysis of ligation products by agarose gel electrophoresis

Ligation efficiency can be assessed by agarose gel electrophoresis of ligation reaction products. For sample loading, usage of SDS-supplemented loading dye, e.g., 6X DNA Loading Dye & SDS Solution (#R1151) is recommended to eliminate band shift due to T4 DNA ligase binding to DNA.

    Prepare the loading mixture:
    Ligation reaction product         10 µl
    6X DNA Loading Dye & SDS Solution (#R1151)（可以自己配）         2 µl
    Heat the sample for 10 min at 65°C and load.

Analysis of ligation reaction products on an agarose gel

ligation reaction         400 ng of vector and insert in total were used. Real ligation experiments normally use less DNA, therefore bands on a gel may appear at lower intensity.
M – GeneRuler™ DNA Ladder Mix (#SM0331).
1 – Mixture of DNA insert and vector in T4 DNA Ligase Buffer.
2 – Mixture of DNA insert and vector after the ligation sample loaded with 6X DNA Loading Dye (#R0611).
3 – Mixture of DNA insert and vector after the ligation sample loaded with 6X DNA Loading Dye & SDS Solution (#R1141).

Interpretation of results


    Appearance of higher molecular weight bands and decreased intensity of the vector and insert bands indicate successful ligation.
    Unchanged band pattern after ligation indicates unsuccessful ligation.