Do you have same experiences? How do you feel if you saw it in your lab?
Showing posts with label Experiment note and tips. Show all posts
Showing posts with label Experiment note and tips. Show all posts
Wednesday, July 25, 2012
Tuesday, July 24, 2012
Hypothesis
Just heard one story concerning the research. One professor has done some trangenic experiment, the purpose is to obtain resistant plant. when she got, it showed really high resistant at the beginning, however, after some days, the plant showed highly infected symptoms. When the student told this results to the supervisor, she did not believe it. Because, in her mind the plant should be resistant and infection only possible caused by contamination. The student has done three individual experiments and got same results, the supervisor still does not believe, because she already think the resistance is the truth, not the results of the experiment....
So, I think it could be really important to have some hypothesis before the research, but it should not affect our mind when we obtain results which does not support the hypothesis. The hypothesis could be right, and could be wrong too.
So, I think it could be really important to have some hypothesis before the research, but it should not affect our mind when we obtain results which does not support the hypothesis. The hypothesis could be right, and could be wrong too.
References............positive and negative
References are important especially when we discuss something,we need some references to support our results, hypothesis, speculations. However, sometime it also could kill the new idea which maybe become milestone in the research.
Friday, April 27, 2012
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
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.
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
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.
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.
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.
Monday, April 23, 2012
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
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.
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.
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