Simply Cloning - Chapter 4 - Gel Purification
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13 سال پیش
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Simply Cloning is a video
Simply Cloning is a video manual for making DNA constructs.
Chapter 4 describes how to separate DNA fragments on agarose gel, and purify them with a gel extraction kit.
Narration Script:
During the gel purification we are going to load the PCR fragment and the digested vector on an agarose gel, apply electric current, and separate DNA molecules based on their molecular weight.
Then we will cut out DNA bands from the agarose and clean them with a gel purification kit.
Gel purification allows us to separate vector DNA from uncut fraction, clean PCR fragment from unspecific PCR products and primers, and also serves as a quality control for the molecular weight of vector and insert.
The summary of gel purification protocol is presented here. Please refer to the Protocols poster for additional details.
Gel purification protocol:
Prepare 1% agarose gel
Add 1 µg of agarose to 100 ml of TEA buffer
Boil in a microwave until the agarose completely dissolved
Add 2 µl ethidium bromide or GelRedTM
Pour into a casting tray
Leave for 30 min to solidify
Load the gel
Molecular weight marker
1 µl of undigested vector
Vector restriction digest
Insert restriction digest
Run the gel until bromophenol blue dye reaches the end
Cut out desired DNA fragments
Purify with a gel purification kit
The advantage of doing vector restriction digest in parallel with PCR is that they both are ready about the same time for gel purification. This way we can run both vector and insert on the same agarose gel.
Let's prepare a 1% agarose gel. I am going to weigh 1.5 grams of agarose and add it to 150 ml of TAE buffer. Then I heat it up in a microwave until it boils.
A safety note: please wear heat-protective gloves when you handle a bottle with boiling agarose. Leave the screw cap on the bottle somewhat loose to allow for vapours to vent.
Then I let the agarose to cool down for 15 min. Add ethidium bromide. Swirl the bottle to mix up ethidium bromide.
Another safety note: remember that ethidium bromide is a cancerogen and change your gloves each time when you get in contact with gels, trays or any other contaminated equipment.
At the moment many laboratories are switching to Gel Red TM (Biotium Inc) - a DNA dye that produces superb DNA staining without being cytotoxic or mutagenic.
Now I am going to pour the agarose in a tray with a comb and let it solidify for 30 minutes.
Finally, I am going to transfer the gel into a running tray and filled with the TAE buffer.
By now both vector restriction digest and the PCR are ready. So, on the agarose gel I am going to run:
Molecular weight marker
1 µl of uncut vector
entire vector restriction digest
and all 50 µl of PCR reaction
To each of these three tubes (except the marker) I have already added 5 ul of the loading dye, which contains bromophenol blue and glycerol.
I am back to the gel room with my tubes and I am ready to load the gel. Once again: molecular weight marker... uncut plasmid... cut plasmid... and the PCR product.
I will run the gel for 35 min at 105 V. Remember, that DNA is negatively charged and therefore it will run towards the positive, or red electrode.
The run is over, let's take the gel to a UV transilluminator and have a look at it.
Here is our DNA separated on the agarose gel. Let me put some labels here. In the left lane we have the marker, then uncut vector, cut vector and the PCR product.
There are few pieces of information that I extract from this kind of picture. First of all, I compare uncut and cut vector lanes. You see how the entire vector band shifted. This means that the vector was linearized.
I also look at the molecular weight of the linearized vector and compare it with the information from the sequence file. The size ofpSAT6-MCS is supposed to be 3900 base pairs, which is in agreement with what I see on the gel.
In addition I roughly estimate the amount of vector DNA. The rule of thumb is that if you can see it well on the gel, then you should have sufficient amount for efficient ligation.
In the case of the PCR product I also look at the molecular weight and at the amount of DNA.
Right now everything looks great, so I will proceed with cutting out the vector and the PCR fragment from the gel.
Before cutting the gel I prepare two labelled Eppendorf tubes, two razor blades, and a UV shield.
Now I am going to put the UV shield on, pull out the transilluminator from the imaging system and turn on UV light.
Chapter 4 describes how to separate DNA fragments on agarose gel, and purify them with a gel extraction kit.
Narration Script:
During the gel purification we are going to load the PCR fragment and the digested vector on an agarose gel, apply electric current, and separate DNA molecules based on their molecular weight.
Then we will cut out DNA bands from the agarose and clean them with a gel purification kit.
Gel purification allows us to separate vector DNA from uncut fraction, clean PCR fragment from unspecific PCR products and primers, and also serves as a quality control for the molecular weight of vector and insert.
The summary of gel purification protocol is presented here. Please refer to the Protocols poster for additional details.
Gel purification protocol:
Prepare 1% agarose gel
Add 1 µg of agarose to 100 ml of TEA buffer
Boil in a microwave until the agarose completely dissolved
Add 2 µl ethidium bromide or GelRedTM
Pour into a casting tray
Leave for 30 min to solidify
Load the gel
Molecular weight marker
1 µl of undigested vector
Vector restriction digest
Insert restriction digest
Run the gel until bromophenol blue dye reaches the end
Cut out desired DNA fragments
Purify with a gel purification kit
The advantage of doing vector restriction digest in parallel with PCR is that they both are ready about the same time for gel purification. This way we can run both vector and insert on the same agarose gel.
Let's prepare a 1% agarose gel. I am going to weigh 1.5 grams of agarose and add it to 150 ml of TAE buffer. Then I heat it up in a microwave until it boils.
A safety note: please wear heat-protective gloves when you handle a bottle with boiling agarose. Leave the screw cap on the bottle somewhat loose to allow for vapours to vent.
Then I let the agarose to cool down for 15 min. Add ethidium bromide. Swirl the bottle to mix up ethidium bromide.
Another safety note: remember that ethidium bromide is a cancerogen and change your gloves each time when you get in contact with gels, trays or any other contaminated equipment.
At the moment many laboratories are switching to Gel Red TM (Biotium Inc) - a DNA dye that produces superb DNA staining without being cytotoxic or mutagenic.
Now I am going to pour the agarose in a tray with a comb and let it solidify for 30 minutes.
Finally, I am going to transfer the gel into a running tray and filled with the TAE buffer.
By now both vector restriction digest and the PCR are ready. So, on the agarose gel I am going to run:
Molecular weight marker
1 µl of uncut vector
entire vector restriction digest
and all 50 µl of PCR reaction
To each of these three tubes (except the marker) I have already added 5 ul of the loading dye, which contains bromophenol blue and glycerol.
I am back to the gel room with my tubes and I am ready to load the gel. Once again: molecular weight marker... uncut plasmid... cut plasmid... and the PCR product.
I will run the gel for 35 min at 105 V. Remember, that DNA is negatively charged and therefore it will run towards the positive, or red electrode.
The run is over, let's take the gel to a UV transilluminator and have a look at it.
Here is our DNA separated on the agarose gel. Let me put some labels here. In the left lane we have the marker, then uncut vector, cut vector and the PCR product.
There are few pieces of information that I extract from this kind of picture. First of all, I compare uncut and cut vector lanes. You see how the entire vector band shifted. This means that the vector was linearized.
I also look at the molecular weight of the linearized vector and compare it with the information from the sequence file. The size ofpSAT6-MCS is supposed to be 3900 base pairs, which is in agreement with what I see on the gel.
In addition I roughly estimate the amount of vector DNA. The rule of thumb is that if you can see it well on the gel, then you should have sufficient amount for efficient ligation.
In the case of the PCR product I also look at the molecular weight and at the amount of DNA.
Right now everything looks great, so I will proceed with cutting out the vector and the PCR fragment from the gel.
Before cutting the gel I prepare two labelled Eppendorf tubes, two razor blades, and a UV shield.
Now I am going to put the UV shield on, pull out the transilluminator from the imaging system and turn on UV light.
13 سال پیش
در تاریخ 1390/12/17 منتشر شده
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