连接产物电泳：来自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.作者: donggua 时间: 2015-7-19 16:57
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作者: donggua 时间: 2015-7-19 16:58
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.作者: donggua 时间: 2015-7-19 16:58
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.作者: donggua 时间: 2015-7-19 16:58
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作者: donggua 时间: 2015-7-19 16:58
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.
作者: donggua 时间: 2015-7-19 16:59
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 作者: donggua 时间: 2015-7-19 17:01
菌落PCR。。。。。。
菌落电泳。。。。。作者: donggua 时间: 2015-7-19 17:01
DNA Electrophoresis(核酸电泳）
1. Low intensity of all or some of the DNA bands （所有泳带不亮，或部分不亮）
1.1. Insufficient amount of ladder was loaded （上样量不足）
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1 mm gel lane width).
1.2. Insufficient or uneven staining （染色不均匀）
Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration 0.5 µg/ml) or SYBR® Green I.
Alternatively, if the DNA will not be used for cloning, add ethidium bromide to both the gel and electrophoresis buffer at a final 0.5 µg/ml concentration.
After alkaline agarose gel electrophoresis the gel should be immersed for 30 min in 300 ml 0.5 M Tris-HCl buffer, pH 7.5 and only later stained in a 0.5 µg/ml ethidium bromide solution for 30 min.
After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15 minutes in 1X TBE to remove the urea prior to staining. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min.
Make sure that the gel is immersed completely in the staining solution.
1.3. DNA run off the gel （核酸飘出上样孔）
Perform electrophoresis until the bromophenol blue dye passes 2/3 (orange G, 4/5) of the gel. Refer to General Recommendations for DNA Electrophoresis for migration of tracking dyes in different gels.
Make sure that the entire gel is immersed completely in the electrophoresis buffer during the run. Make sure that gel and apparatus are positioned horizontally during the run.
1.4. DNA diffusion in the gel （核酸已经在胶里面扩散）
Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion of smaller DNA fragments in the gel.
Avoid long term storage of the gel before taking a picture, as this may cause diffusion of DNA fragments and low band intensity.
1.5. DNA masking by electrophoresis tracking dyes（颜色指示剂掩盖dna.)
Do not exceed the amount of electrophoresis tracking dyes used for sample/ladder preparation. Use the loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions contain equilibrated amount of tracking dyes which will not mask DNA under UV light.
Prepare DNA ladders and probes according to recommendations.作者: donggua 时间: 2015-7-19 17:01
核酸电泳 Smeared DNA bands (弥散带）
2.1. DNA degradation by nucleases
Use fresh electrophoresis buffers, freshly poured gels, nuclease free vials and tips to minimize nuclease contamination of DNA solutions.
2.2. Improper electrophoresis conditions
Prepare gels according to recommendations, always use the same electrophoresis buffer for both preparation of the gel and running buffer.
Make sure that the whole gel is immersed completely in the electrophoresis buffer during the run.
Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To increase the band sharpness, use a lower voltage for several minutes at the beginning of electrophoresis.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O'GeneRuler™ Express DNA ladders.
An excessively low voltage during the entire run may result in diffusion of bands during electrophoresis. Excessively high voltage may result in gel heating and DNA denaturation.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (usually 5-8 V/cm, depending on the ladder) one has to:
measure the distance between electrodes (cathode and anode) – X, cm,
and multiply that X, cm value by the recommended voltage (Y, V/cm),
the result (X, cm x recommended Y, V/cm) is Z – recommended voltage to be applied.
2.3. Gel shift effect
DNA binding proteins, such as ligases, phosphatases or restriction enzymes may alter DNA migration on gels and cause the DNA to remain in the gel well or gel shifting.
Lambda DNA or other DNA with long complementary overhangs may anneal and migrate atypically.
To correct for the above mentioned effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.
2.4. Excess DNA loaded
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1 mm gel lane width). If possible apply same requirements for DNA quantities for the samples as well.
2.5. High salt concentration in the sample
Samples containing high concentrations of salts may result in smeared or shifted band patterns.
Ethanol precipitation and washing the pellet with ice cold 75% ethanol or spin column purification prior to resuspending the sample in water or TE buffer helps eliminate saltspresent in the sample.
2.6. Poorly formed (slanted) gel wells
When inserting the comb into the gel, make sure that it is vertical to the gel surface and stable during gel casting and its solidification.作者: donggua 时间: 2015-7-19 17:02
3. Atypical banding pattern
3.1. Lambda DNA marker was not heated prior to loading
All DNA markers generated from Lambda DNA, as well as lambda DNA digestion products should be heated at 65°C for 5 min and chilled on ice before loading on the gel in order to completely denature the cohesive ends (the 12 nt cos site of lambda DNA) that may anneal and form additional bands.
3.2. Denatured DNA
Excessively high voltage may result in gel heating and DNA denaturation.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (often 5-8 V/cm, depending on the ladder) one has to:
measure the distance between electrodes (cathode and anode) – X, cm,
and multiply that X, cm value by the recommended voltage (Y, V/cm),
the result (X, cm x recommended Y, V/cm) is Z – recommended voltage to be applied.
For non-denaturing electrophoresis use the loading dye solutions supplied with every Fermentas DNA ladder/marker, as these solutions do not contain denaturing agents.
Prepare DNA ladders and probes according to recommendations.
Do not heat them before loading. Heating is required only for lambda DNA markers.
3.3. Different loading conditions for the sample and the ladder DNA
Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
If possible always load equal or very similar volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted with 1X loading dye.
3.4. Improper electrophoresis conditions
Excessive electrophoresis run times or voltage may result in migration of small DNA fragments off of the gel. Very short or slow electrophoresis may result in incompletely resolved bands.
Run gels at 5-8 V/cm until the bromophenol blue passes 2/3 (orange G, 4/5) of the gel. Refer to General Recommendations for DNA Electrophoresis for migration of tracking dyes in different gels.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O'GeneRuler™ Express DNA ladders.
3.5. Incorrect gel percentage or running buffer used
TAE buffer is recommended for analysis of DNA fragments larger than 1500 bp and for supercoiled DNA. TBE buffer is used for DNA fragments smaller tha 1500 bp and for denaturing polyacrylamide gel electrophoresis. Large DNA fragments will not separate well in TBE buffer.
The correct gel percentage is important for optimal separation of the ladder DNA; prepare gels according to recommendations. When preparing agarose gels always adjust the volume of water to accommodate for evaporation during boiling. Otherwise, the gel percentage will be too high and result in bad separation of larger DNA bands.
Refer to General Recommendations for DNA Electrophoresis for the range of effective separation of DNA in different gels.
Ethidium bromide interferes with separation of large DNA fragments. Do not include ethidium bromide in the gel and run buffer when large DNA (more than 20 kb) or supercoiled DNA is analyzed. Stain the gel following electrophoresis in a 0.5 µg/ml ethidium bromide solution for 30 min.
3.6. Atypical migration due to different DNA sequence or structure
During high resolution electrophoresis DNA fragments of equal size can migrate differently due to differences in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. The sequences of Fermentas DNA ladders are chosen to allow for highly accurate DNA migration according to size, however, due to differencies in nucleotide sequence or the overall DNA structure, sample migration can sometimes slightly differ from ladder band migration.
DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels compared to an equivalent DNA size standard. See the picture below for migration of plasmid DNA forms:
Figure. Migration of plasmid DNA forms
GeneRuler™ 1 kb DNA Ladder
Undigested plasmid pUC19 DNA (2,7 kb), forms:
upper band (~4 kb) – dimeric plasmid,
below, less visible (~3.5 kb) – nicked plasmid,
lowest band (~1.9 kb) – supercoiled plasmid.
Linearized plasmid pUC19 (2,7 kb) – migrates according to its size.
High level DNA modifications such as methylation, labeling with biotin or large fluorescent molecules also result in slower migration compared to unmodified DNA of the same size.
3.7. Gel shift effect
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel or cause the DNA to remain in the gel wells.
Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical migration pattern.
To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.
High salt concentration in the sample may also cause gel shift effects, see 3.8.
3.8. High salt concentration in the sample
Samples with a high salt concentration may give smeared or shifted band patterns.
Ethanol precipitation and washing the pellet with ice cold 75% ethanol or spin column purification prior resuspending DNA in water or TE buffer, helps eliminate salt from the sample.作者: donggua 时间: 2015-7-19 17:02
Curved DNA bands
4.1. Gel incompletely immersed in electrophoresis buffer
Electrophoresis buffer should completely cover the entire gel during sample loading and run.
4.2. Low sample volume
The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large wells should not be used with small sample volumes. If needed the sample volume can be adjusted with 1X loading dye.
4.3. Improper electrophoresis conditions
Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band curving, use a lower voltage for several minutes at the beginning of electrophoresis.
For fast electrophoresis under high voltage (up to 23 V/cm) use GeneRuler™ or O'GeneRuler™ Express DNA ladders.
To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which is in many cases 5-8 V/cm, depending on the ladder) one has to:
measure the distance between electrodes (cathode and anode) – X, cm,
and multiply the X value by the recommended voltage (Y, V/cm),
the result (X x Y) is the recommended voltage to be applied.
4.4. Bubbles or physical particles in the gel wells or in the gel
Use pure water, clean flasks and clean equipment for preparation of gels.
Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.作者: donggua 时间: 2015-7-19 17:02
DNA remains in the gel
5.1. Poorly formed gel wells
Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately.
Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample.
5.2. Excess DNA loaded
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1mm gel lane width). If possible load the same quantity of the sample.
5.3. Contamination of the DNA sample
Make sure that your sample DNA solution does not contain any precipitate.
5.4. Gel shift effect
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel and cause the DNA to remain in the gel wells.
Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band migration pattern.
To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends.
Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load.作者: donggua 时间: 2015-7-19 17:03
