Team:Bielefeld-CeBiTec/Results/Library/Assembly
Library Results
Assembly
Assembly
We annealed v1.1+v1.2 to v1 and v2.1+v2.2 to v2, and then v1+v2 to the variable region for the Monobody construct. To get further insight take a look here.
The size of the annealed v1 fragment is ought to be 114 bp, the size of v2 is 132 bp. This then can be inserted into our Monobody construct (BBa_K2082000).
We annealed F2.1 and F2.2 to the region containing the variable region called F2 for the Nanobody construct. To get further insight take a look here.
The size of the annealed F2 fragment is ought to be 143 bp. This then can be inserted into our Nanobody construct (BBa_K2082001)
The size of the annealed v1 fragment is ought to be 114 bp, the size of v2 is 132 bp. This then can be inserted into our Monobody construct (BBa_K2082000).
We annealed F2.1 and F2.2 to the region containing the variable region called F2 for the Nanobody construct. To get further insight take a look here.
The size of the annealed F2 fragment is ought to be 143 bp. This then can be inserted into our Nanobody construct (BBa_K2082001)
Comparison of Polymerases
As our variable regions (for each Monobodies and Nanobodies) were ordered through oligonucleotide synthesis, the strands naturally were single stranded. For the correct assembly of the variable regions into the construct, they had to be annealed. For this purpose we tried using the Klenow Fragment (3'→5' exo-) by New England Biolabs at first. However, the cloning of the Klenow annealed fragments was not optimal.
For example, when inserting the variable regions for the Nanobodies, the primers called F2.1 and F2.2 had to be annealed, to then be cloned into the Nanobody construct (missing the variable regions). When separating the samples (annealed oligonucleotides) on gel the reason why was hinted as no fragments of 143 bp were found in the bands on which the annealed sample were loaded (see Figure 1).
For example, when inserting the variable regions for the Nanobodies, the primers called F2.1 and F2.2 had to be annealed, to then be cloned into the Nanobody construct (missing the variable regions). When separating the samples (annealed oligonucleotides) on gel the reason why was hinted as no fragments of 143 bp were found in the bands on which the annealed sample were loaded (see Figure 1).
Figure 1: Gelelectrophoresis of annealed oligonucleotides. Photography of 1% agarose gel dyed with ethidium bromide. Loading of the samples (from right to left): Primer F2.1, Primer F2.2, 100bp plus ladder (by New England Biolabs), empty space, three times the by the Klenow Fragment annealed parts, empty space, 100bp plus ladder (by New England Biolabs).
We then concluded to use other Polymerases to anneal the oligonucleotides. For that we annealed two oligonucleotides called v1.1 and v1.2 to a fragment called v1 as well as v2.1 and v2.2 to a fragment called v2. These were used to bring in the variable region of the Monobody construct, inhibiting the randomized regions essential for binding proteins in the following.
For this purpose we used the following polymerases: Q5 High-Fidelity DNA Polymerase (by New England Biolabs), KOD DNA Polymerase (by Merck Millipore), Phusion High-Fidelity DNA Polymerase (by New England Biolabs), GoTaq G2 (by Promega).
In Figure 2.1 one can see the result of the annealing of the oligonucleotides v1.1 and v1.2 to the fragment v1 and v2.1 and v2.2 to the fragment v2 by the four polymerases.
Figure 2.1: Gelelectrophoresis of annealed oligonucleotides. Photography of 1% agarose gel dyed with ethidium bromide. Loading of the samples (from right to left): Low Molecular Weight DNA Ladder (by New England Biolabs), Fragment v1 annealed with GoTaq G2 (by Promega), Fragment v1 annealed with KOD DNA Polymerase (by Merck Millipore), Fragment v1 annealed with Phusion High-Fidelity DNA Polymerase (by New England Biolabs), Fragment v1 annealed with Q5 High-Fidelity DNA Polymerase (by New England Biolabs), Fragment v2 annealed with GoTaq G2 (by Promega), Fragment v2 annealed with KOD DNA Polymerase (by Merck Millipore), Fragment v2 annealed with Phusion High-Fidelity DNA Polymerase (by New England Biolabs), Fragment v2 annealed with Q5 High-Fidelity DNA Polymerase (by New England Biolabs).
This was also used with the same four Polymerases mentioned above for the annealing of the oligonucleotides F2.1 and F2.2 to fragment F2 containing the variable region for the Nanobody. The gel photography can be viewed in Figure 2.2.
After choosing the Q5 High-Fidelity DNA Polymerase (by New England Biolabs) for the annealing of the oligonucleotides we once again checked for the right size of the fragments mentioned above (v1, v2 and F2) on an 1% agarose gel (see Figure 3).
We so concluded to proceed with further annealing with the Q5 High-Fidelity DNA Polymerase (by New England Biolabs). This also resulted in a greater number of clones counted in later cloning.
