Cloning
If you want to do a genetic analysis of a specific gene for a longer period, you have three options
1. Do DNA-extraction from a specific tissue - PCR to amplify the GOI
a. But each times, the DNA molecules are needed
2. Do the DNA-extraction once, and amplify the GOI over and over again using PCR
a. But there will start appearing mutations in your amplicon (no polymerase with proofreading)
b. PCR product cant be stored for a longer period
3. Do the DNA-extraction once, followed by a single PCR
the obtained amplicon will be ligated in a plasmid that will be transformed into a bacterial cell
a. These cells do have polymerases with proofreading (overcomes mutations)
b. Can be stored for many years at -80°C
c. The plasmid can be multiplied very easy over generations
d. Via a method called miniprep, the plasmids can be purified quickly from the bacteria
--- first session ---
We are doing pGEM-T cloning = TA-cloning
▪ Refers to a plasmid (or vector) in where we are going to put our GOI after amplification during PCR → pGEM-T easy vector
▪ The pGEM-T vector is naturally linear which is weird cause bacteria can only handle circular plasmids
▪ The linear pGEM-T vector has 2 overhanging T ends
o Because of this, the right primers need to be used in the PCR reaction so the GOI can be amplified and also has 2
overhanging ends (A now)
o That is how the GOI with its two overhanging A’s can be inserted in the linear vector with two overhanging T’s
via T4-ligase and because of this, the plasmid rolls up and becomes circular (functional)
▪ After this, we are going to add the vector in E. Coli cells via heat shock transformation
o You’ll get tiny pores in the bacterial cell wall (42°C)
o By adding the plasmid to the cells, then heat shocking, the plasmids will enter the cell
o Butt the cell need to be made heat shock competent via freezing in a buffer with Ca2+
▪ Because DNA and the cell wall are both negatively charged so they would repel each other
▪ The cell wall will be positively charged and that’s how the transformation will be facilitated
o After this cooling so the cell wall closes again
Intermezzo
- Cloning = volledige process waarbij je GOI in vector steek ten laat vermeerderen in bacteriën
o GOI → PCR → in plasmide ligeren (ligation) → in bacterie brengen (transformation)
- Ligeren = de chemische “plakstap”
o Hebt een geknipt plasmide → in de multiple cloning site (MCS) geknipt door restrictive-enzymen
o Hebt PCR product
- Transforamtion = het binnenbrengen van het plasmide in abcteriën
o Meestal via heat-shock
, Selection 1: Now we want to check if the transformation has worked, cause some won’t have inserted the plasmid (selection)
▪ Plate bacteria on LB medium with the antibiotic carbenicillin
▪ The pGEM-T plasmid has a gene that codes for a protein that can degrade carbenicillin
o So if the transformation was successful, the cell will have the plasmid and can grow into a colony
o Also cells that incorporated a plasmid that’s still linear (no PCR product in → circular) wont survive either cause
bacteria cant handle linear plasmids
▪ Important: you need some time between plating them out after the heat shock cause the cells need time to transcribe the
gene and produce the antibiotic resistance → (30min 37°C)
▪ Incubation overnight to grow individual colonies
--- second session ---
Selection 2: Blue-white screening (using the lacZgen as a hallmark of pGEM-T cloning)
▪ Its possible that a cell contains a self-ligated plasmid:
o During the ligation, it’s possible that the T4-ligase closes a linear plasmid without insertion of the GOI
▪ In theory impossible because of the two T-ends, but in practise often observed
o They will also live cause they also have the antibiotic resistance gene
▪ On both ends of the linear plasmid are parts of the lacZgen
o When it self-ligates and that plasmid transforms into a cell, this lacZgen will be functional
o It can produce 𝜷-galactosidase that can degrade lactose or X-gal
o When X-gal is cleavaged, is leads to a blue chemical
o Therefore: X-gal will be also added to the solid medium before incubation, after you ll notice
▪ White colonies: they have to correctly ligated plasmid with the GOI
▪ Blue colonies: they have a self-ligated plasmid
▪ Rest: died cause they don’t have a plasmid and therefore no antibiotic resistance
▪ When the GOI is inserted into the plasmid (at both ends to make it circular) the lacZgen is not functional
Now you can pick up a white colony and further cultivate it in a liquid LB
--- third session ---
Then a miniprep will be done in which the plasmid will be selectively purified out of the bacteria
Since we only have cells left which contain the plasmid with the GOI, and a lot of cells, we can obtain a very high
number of plasmids
3 main steps
1. Making the DNA available (and maintaining it)
a. Adding NaOH and SDS buffer
i. SDS = detergent which destabilizes the membrane, at high T, it cause cell lysis
ii. SDS = denaturing agent which breaks down the 3D structure of proteins
→ it binds on proteins, unfolds them and covers their charges, so the result is a negative charge
b. Adding EDTA-containing buffer
i. EDTA binds Mg2+, which is very important
ii. Cause DNAse enzymes, which cut DNA, are normally controlled via control pathways in the cell but upon cell
lysis, these control pathways disappear, so they would start cutting the genomic plasmids and our vector
iii. Mg2+ is an essential co-factor for DNAse enzymes so it gets taken away by EDTA
If you want to do a genetic analysis of a specific gene for a longer period, you have three options
1. Do DNA-extraction from a specific tissue - PCR to amplify the GOI
a. But each times, the DNA molecules are needed
2. Do the DNA-extraction once, and amplify the GOI over and over again using PCR
a. But there will start appearing mutations in your amplicon (no polymerase with proofreading)
b. PCR product cant be stored for a longer period
3. Do the DNA-extraction once, followed by a single PCR
the obtained amplicon will be ligated in a plasmid that will be transformed into a bacterial cell
a. These cells do have polymerases with proofreading (overcomes mutations)
b. Can be stored for many years at -80°C
c. The plasmid can be multiplied very easy over generations
d. Via a method called miniprep, the plasmids can be purified quickly from the bacteria
--- first session ---
We are doing pGEM-T cloning = TA-cloning
▪ Refers to a plasmid (or vector) in where we are going to put our GOI after amplification during PCR → pGEM-T easy vector
▪ The pGEM-T vector is naturally linear which is weird cause bacteria can only handle circular plasmids
▪ The linear pGEM-T vector has 2 overhanging T ends
o Because of this, the right primers need to be used in the PCR reaction so the GOI can be amplified and also has 2
overhanging ends (A now)
o That is how the GOI with its two overhanging A’s can be inserted in the linear vector with two overhanging T’s
via T4-ligase and because of this, the plasmid rolls up and becomes circular (functional)
▪ After this, we are going to add the vector in E. Coli cells via heat shock transformation
o You’ll get tiny pores in the bacterial cell wall (42°C)
o By adding the plasmid to the cells, then heat shocking, the plasmids will enter the cell
o Butt the cell need to be made heat shock competent via freezing in a buffer with Ca2+
▪ Because DNA and the cell wall are both negatively charged so they would repel each other
▪ The cell wall will be positively charged and that’s how the transformation will be facilitated
o After this cooling so the cell wall closes again
Intermezzo
- Cloning = volledige process waarbij je GOI in vector steek ten laat vermeerderen in bacteriën
o GOI → PCR → in plasmide ligeren (ligation) → in bacterie brengen (transformation)
- Ligeren = de chemische “plakstap”
o Hebt een geknipt plasmide → in de multiple cloning site (MCS) geknipt door restrictive-enzymen
o Hebt PCR product
- Transforamtion = het binnenbrengen van het plasmide in abcteriën
o Meestal via heat-shock
, Selection 1: Now we want to check if the transformation has worked, cause some won’t have inserted the plasmid (selection)
▪ Plate bacteria on LB medium with the antibiotic carbenicillin
▪ The pGEM-T plasmid has a gene that codes for a protein that can degrade carbenicillin
o So if the transformation was successful, the cell will have the plasmid and can grow into a colony
o Also cells that incorporated a plasmid that’s still linear (no PCR product in → circular) wont survive either cause
bacteria cant handle linear plasmids
▪ Important: you need some time between plating them out after the heat shock cause the cells need time to transcribe the
gene and produce the antibiotic resistance → (30min 37°C)
▪ Incubation overnight to grow individual colonies
--- second session ---
Selection 2: Blue-white screening (using the lacZgen as a hallmark of pGEM-T cloning)
▪ Its possible that a cell contains a self-ligated plasmid:
o During the ligation, it’s possible that the T4-ligase closes a linear plasmid without insertion of the GOI
▪ In theory impossible because of the two T-ends, but in practise often observed
o They will also live cause they also have the antibiotic resistance gene
▪ On both ends of the linear plasmid are parts of the lacZgen
o When it self-ligates and that plasmid transforms into a cell, this lacZgen will be functional
o It can produce 𝜷-galactosidase that can degrade lactose or X-gal
o When X-gal is cleavaged, is leads to a blue chemical
o Therefore: X-gal will be also added to the solid medium before incubation, after you ll notice
▪ White colonies: they have to correctly ligated plasmid with the GOI
▪ Blue colonies: they have a self-ligated plasmid
▪ Rest: died cause they don’t have a plasmid and therefore no antibiotic resistance
▪ When the GOI is inserted into the plasmid (at both ends to make it circular) the lacZgen is not functional
Now you can pick up a white colony and further cultivate it in a liquid LB
--- third session ---
Then a miniprep will be done in which the plasmid will be selectively purified out of the bacteria
Since we only have cells left which contain the plasmid with the GOI, and a lot of cells, we can obtain a very high
number of plasmids
3 main steps
1. Making the DNA available (and maintaining it)
a. Adding NaOH and SDS buffer
i. SDS = detergent which destabilizes the membrane, at high T, it cause cell lysis
ii. SDS = denaturing agent which breaks down the 3D structure of proteins
→ it binds on proteins, unfolds them and covers their charges, so the result is a negative charge
b. Adding EDTA-containing buffer
i. EDTA binds Mg2+, which is very important
ii. Cause DNAse enzymes, which cut DNA, are normally controlled via control pathways in the cell but upon cell
lysis, these control pathways disappear, so they would start cutting the genomic plasmids and our vector
iii. Mg2+ is an essential co-factor for DNAse enzymes so it gets taken away by EDTA
