For transformation of bacteria cells, in this study the method of heat shock was applied.
Therefore, 100 µl of a suspension of competent E.Coli TOP10 (see 8.6.1) were thawed on ice. The plasmid (10-50 ng) was added and carefully mixed with a pipette tip. After incubation on ice for 30 min, the bacteria were exposed to the heat shock: the tube containing the bacteria suspension was incubated at 37 °C in a water bath for 2 min, followed by two minutes incubation on ice. Subsequently, 200 µl of LB medium (without antibiotics) were added and carefully mixed with a pipette tip. The suspension was incubated in a thermal block at 300 rpm, 37 °C for 1 h, before it was spread on a LB agar plate with ampicillin (100 µg/ml), which was incubated at 37 °C over night.
Since these agar plates contained ampicillin, only successfully transformed bacteria possessing recombinant plasmids with an ampicillin resistance gene survived.
8.6.3 Cultivation of bacteria
Bacteria (e.g. a selected colony from an agar plate or an aliquot of a glycerol stock) were given into 4 ml LB medium with ampicillin (100 µg/ml). The suspension was incubated over night in a bacteria shaker (220 rpm, 37 °C).
8.6.4 Plasmid isolation
One of the most frequently used methods to isolate plasmid DNA from bacteria is based on the principle of alkaline lysis.236 Therefore, bacteria containing the plasmid of interest are first centrifuged and obtained as a pellet. This pellet is resuspended in a specific buffer containing NaOH and SDS, which cause lysis of the cells as well as denaturation of the DNA, RNA and proteins due to the high pH level. RNA is degraded in the presence of RNAse. In the presence of EDTA divalent cations are complexed and thus removed from the environment, whereby bacterial nucleases are hindered in their function to degrade plasmid DNA. Subsequently, an acetic acid/acetate buffer is added, which neutralizes the pH level. In this milieu, small plasmid DNA renaturates and passes into solution, while genomic bacterial DNA only renaturates incompletely and remains precipitated as do proteins and other cellular components. By centrifugation plasmid DNA can then be separated from the precipitated material. Adsorption of plasmid DNA to silica membrane columns allows purification of the DNA with ethanol based washing buffers and a final elution with pure H2O.
Isolation of plasmid DNA in this study was performed with different kits (see 8.1.3.3) according to the manufacturer’s protocol. These kits are all based on the above described principle.
8.6.5 Determination of DNA concentration
A 1:500 dilution (2 µl DNA solution + 998 µl water) of the DNA solution was prepared to determine the concentration photometrically. The measurement was made at 260 nm using water as a blank.
8.6.6 Preparation of glycerol stocks
For long-term storage of bacteria, an aliquot of the culture was conserved as glycerol stock. Therefore, 800 µl of a bacteria culture were mixed with 200 µl glycerol and stored at -20 °C. To recultivate bacteria, a small amount of the glycerol culture was given into LB medium with ampicillin (100 µg/ml) and incubated in the bacteria shaker (220 rpm, 37 °C) over night.
8.6.7 Primer design
All primers applied in this study were either designed as primer for PCR or for DNA sequencing reactions (see 8.1.3.3). What they all have in common, is the requirement to bind as specific as possible to a certain sequence of the DNA. Only that way it can be guaranteed that the DNA polymerase, which uses the primer as starter oligonucleotide for elongation of the complementary strand, will exclusively amplify the DNA sequence of interest. For the performance of a PCR as well as a sequencing reaction, a pair of primers had to be designed (forward and reverse), which are complementary to the 3’ end of the sense and anti-sense strand and therewith flank the DNA sequence of interest. Ideally, a primer possesses a GC-content (guanine-cytosine content) in a range of 45 to 60%, a melting temperature between 55 and 70 °C and comprises a length of 18 to 22 nucleotides. Furthermore, it should not form any stable hairpins (intramolecular base pairing), nor stable dimers with other primer molecules (intermolecular base pairing). Primers designed for this study have been analyzed with the online program Oligoanalyzer 3.1, Integrated DNA Technologies (see 8.1.1).
8.6.8 Polymerase chain reaction
The polymerase chain reaction (PCR), which was invented by Kary Mullis in 1983 (nobel prize 1993), is an efficient and fast method for the amplification of DNA. It is based on a heat program, which – depending on the present temperature – causes denaturation or renaturation of the DNA template. In the presence of a heat stable DNA polymerase, DNA strands are amplified by elongation in 5’- to 3’-direction. Therefore, a pair of specific primers (see 8.1.3.3) is needed, which flank the template sequence and serve as starter oligonucleotides for the polymerase. As building blocks for the emerging strand the polymerase requires nucleotides of the four bases A, T, G and C, which are added in form of dNTPs (ATP, TTP, GTP and CTP) into the reaction mixture. It is further important to choose an appropriate buffer system with additions like e.g. certain cations, providing an optimized working environment for the polymerase.
In a typical PCR, the reaction mixture, which is placed in a thermocycler, is first heated up to a temperature that lies above the melting temperature (Tm) of the DNA strand.
This step (e.g. 94 °C for a couple of seconds) causes the double-stranded DNA to denaturate into two single strands. Subsequently, the thermocycler cools down to a temperature that is just a few degrees beneath the melting temperature (Tm) of the primers. The temperature, which roughly lies between 55 and 65 °C, is dependent on the GC-content of the primers. During this second step the primers anneal to the 3’ end of the sense or anti-sense strand, respectively. Afterwards, the elongation of the DNA strand is achieved by heating up to a temperature (e.g. 72 °C) that represents the temperature optimum for the DNA polymerase. The duration of the third step is dependent on how quickly the polymerase can work under the given conditions (e.g.
proceeding speed of 1 kbp/min) and should be modified according to the template’s size. This three-step cycle (denaturation, annealing, elongation) is repeated several times (typically 30 to 35 cycles), which causes an exponential amplification of the DNA template.
In this study the PCR protocols listed in the following were used. Annealing temperatures (Tann.) were chosen based on the Tm of the applied primers. The duration of the elongation step was determined by the size of the template and the proceeding speed of the polymerase.
Table 24: PCR with PyrobestTM DNA polymerase.
PCR mixture temperature program
x µl template DNA 20 ng
2 µl f-primer 10 pmol
30 x
98 °C 10 s denaturation
2 µl r-primer 10 pmol Tann. 30 s annealing
0.5 µl PyrobestTM DNA Polymerase 72 °C 1 kbp/min elongation 5 µl 10x PyrobestTM Buffer II
4 µl dNTPs Mixture (2.5 mM) 72 °C 10 min final
elongation ad 50 µl H2O, sterile
Table 25: PCR mit AccuPrimeTM Pfx DNA polymerase.
PCR mixture temperature program
95 °C 2 min initial
denaturation
x µl template DNA 20 ng
2 µl f-primer 10 pmol
35 x
95 °C 15 s denaturation
2 µl r-primer 10 pmol Tann. 30 s annealing
0.8 µl Accu PrimeTM Pfx DNA Polymerase 68 °C 1 kbp/min elongation 5 µl 10x Accu PrimeTM Pfx DNA mix
ad 50 µl H2O, sterile 68 °C 5 min final
elongation
Table 26: KOD Hot Start DNA polymerase.
PCR mixture temperature program
x µl template DNA 20 ng
3 µl f-primer 15 pmol 94 °C 2 min initial
denaturation
3 µl r-primer 15 pmol
1 µl KOD Hot Start DNA polymerase
35 x
94 °C 20 s denaturation
5 µl 10x buffer Tann. 15 s annealing
5 µl dNTPs Mixture (2 mM) 70 °C 3 kbp/min elongation
4 µl MgSO4 (25 mM)
5 µl DMSO 70 °C 10 min final
elongation ad 50 µl H2O, sterile
In this study, after the PCR has been completed, the reaction mixture was mixed with 6x loading dye (see 8.1.3.3 and 8.1.3.6, 1 µl per final volume of 6 µl) and loaded onto an agarose gel (see 8.6.9) for purification.
8.6.9 Agarose gel electrophoresis
Agarose gel electrophoresis is a simple method for the separation of nucleic acids using a gel, whose pore size can be varied by the concentration of added agarose. For separation, the samples are given into small wells on the upper end of the gel placed in TAE buffer (pH ~ 8). When subjected to an electrical voltage, the negatively charged nucleic acid molecules migrate in the electrical field towards the anode. Short and small molecules migrate faster and therewith run further in the gel as do long and bulky molecules, which are stronger retained by the gel matrix. By addition of an intercalating dye (e.g. ethidium bromide) into the gel, it is possible to visualize the nucleic acids by light emission under UV light afterwards.
Within this study 1% agarose gels were used, for which agarose was given into TAE buffer (e.g. 500 mg in 50 ml) and carefully heated in the microwave until agarose had completely dissolved. After short cooling-down, the intercalation dye (here GelRed®) was added (1:20000 dilution, here 2.5 µl) and homogeneously distributed. The gel was poured into a gel chamber and after hardening, was transferred to the electrophoresis chamber filled with TAE buffer. 6x loading dye (see 8.1.3.3 and 8.1.3.6) was added to the DNA samples as well as to the respective DNA ladder (molecular weight size marker, see 8.1.3.3) and each sample was transferred into a well of the gel. The electrophoresis was run at 100 to 200 V depending on the size of the electrophoresis chamber. Subsequently, the gels were analyzed under UV light.
8.6.10 Gel extraction
After separation by gel electrophoresis, the band of interest was cut out of the agarose gel. From this piece of gel the DNA was regained using the ZymocleanTM Gel DNA Recovery kit according to the manufacturer’s protocol. Thereby, the agarose gel is solubilized in a high-salt binding buffer liberating the DNA, which afterwards is purified on small silica columns.
8.6.11 Restriction enzyme digestion
Restriction endonucleases are enzymes that recognize certain sequences on the DNA molecule and thereupon cleave the strands in an enzyme specific manner. In this study for restriction endonuclease digestion, enzymes from New England BioLabs (see 8.1.3.3) were used in combination with supplied buffers and additives according to the manufacturer’s protocol. If two enzymes were used that needed different reaction conditions the digestion was performed sequentially. In general, the following protocol was used:
DNA (plasmid or PCR product) x µl reaction buffer (10x) 1 µl
restriction enzymes 10 U, each
(BSA solution 10x, if needed) 1 µl
H2O, sterile ad 10 µl
If not indicated otherwise in the manufacturer’s protocol, after incubation at 37 °C for 1 h, the enzymes were heat inactivated by incubation at 65 °C for 20 min. The digested product was either purified by agarose gel electrophoresis (see 8.6.9) or – as was the case for digested PCR products – using the DNA clean & concentratorTM-5 kit (see 8.1.3.3).
For plasmid DNA that was linearized in order to be applied for stable transfections (see 8.7.6.1, c and d), 50 µg of the plasmid was cleaved using 50 U of the below mentioned restriction enzyme in the presence of 1x BSA at 37 °C for 3 h.
pQCXIH-hSV2A-GFP: FspI NEbuffer 4 pQCXIN-hGluR2flip: StuI NEbuffer 4 pQCXIH-hGluR2flop: FspI NEbuffer 4
8.6.12 Ligation
For ligation of two nucleic acid sequences (e.g. a digested PCR product with a digested plasmid), the ATP-dependent enzyme ligase, was used. This enzyme ligates the 3’-hydroxy terminus of one fragment with the 5’-phosphate terminus of the other fragment under formation of a phosphodiester bond. The reaction mixture was prepared according to the following protocol:
plasmid, digested 50 ng PCR product, digested 150 ng 10 x ligation buffer 1 µl
T4 DNA ligase 2 U
ATP (10 mM) 1 µl
H2O, steril ad 10 µl
The ligation mixture was incubated at 16 °C over night and afterwards directly used for transformation of competent bacteria (see 8.6.2).
8.6.13 Sequencing
Analysis of DNA sequences was performed by GATC Biotech AG, Konstanz.