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4.2 Results
4.2.4 Further peR reactions tested using various primers designed
4.2.6.5 Screening for recombinant pGEX plasmids using DIG nucleic acid detection
Insert DNA can be directly detected from DNA prepared from a single colony,and so allows for rapid screening of multiple colonies on a transformation plate. As a control for this screening technique,an oligonucleotide probe was constructed specific to pGEX vector DNA, independent of insert DNA. As described in Section 2.2.1, universal pGEX PCR and sequencing primer pG-Seq1 (GGG CTG GCA AGC CAC GTT TGG TG) was tailed at the 3' terminus with the DIG-ddUTP compound. Serial dilutions of purified pGEX-4T-3 plasmid
resulting from transformation with the pGEX (non-recombinant) vector. The membrane was probed using the DIG-labelled oligonucleotide, specific to pGEX vector DNA, to access the degree of detection and sensitivity of this technique. Figure 4.10 shows the nylon membrane with these samples, detected with anti-DIG antibody conjugated with alkaline phosphatase and,and detected with the substrates NBT and BeIP (Section 2.2.2).
2 3
4 5 6 7 8 9 10
G)
Figure 4.10 DIG nucleic acid detection of DNA from colonies using pGEX specific probe. 1 to 3 - 5 ul DNA prepared from single colonies of pGEX;4 to 10 - 5 ul of the serial dilution of purified vector DNA (1000 ng; 500 ng; 250 ng; 125 ng; 62.5 ng; 31.25 ng; 15.125 ng).
As can be seen in Figure 4.10, the colour reaction is more intense with a higher concentration of DNA and gradually diminishes as the concentration is decreased sequentially. The crude DNA preparation from the pGEX colonies showed positive colour reactions, and although not as intense, is still visible against the background as a positive reaction. In comparison to the serial dilution, the intensity of the colour reaction is similar to that of number 8, 9 and 10.
This indicates that the plasmid DNA in each colony is present in the approximate range of 15,125 - 62.5 ng. This result also shows that the screening can be performed in the same manner, but replacing the vector specific probe with insert specific DIG-labelled oligonucleotide,for the detection of insert DNA.
The oligonucleotide primer PfFOR2 (BamHI) used for the amplification of the 817 bp region (Section 4.2.3) was tailed with DIG, and used as a probe for the detection of insert DNA in colonies. A number of colonies were screened as shown in Figure 4.11. The control for the immunochemical detection of DIG and the alkaline phosphatase.colour reaction was DIG- labelled molecular weight markers. The hybridisation reaction was checked using the purified
colour reaction, indicating that the hybridisation reaction was working efficiently, and the synthesised probe is specific to the 817 bp insert.
Figure 4.11 Screening by DIG nucleic acid detection using insert specific DIG-labelled oligonucleotide probe. Cl - DIG labelled molecular weight markers; C2 - 817 bp insert DNA;C3 - pGEX (non-recombinant) DNA;1 to 47 - crude DNA preps from 47 colonies.
No colour reaction was detected for the pGEX control sample dotted, indicating that no false positives were likely to occur due to non-specific hybridisation. There were no positive colour reactions seen for any of the colonies screened. This experiment was repeated for a further 256 colonies,with no success in identifying a positive clone.Due to the fact that this method of screening, allowed a great number of clones to be screened,it was assumedthatthe ligation
. .
reactions with pGEX did not result in recombinants, A more efficient system of cloning the 817 bp insert was.attempted, using the pMOSBlue blunt-end cloning kit, for initially obtaining a vector containing the insert DNA attempted in these experiments ..
4.2.7 Transformation of competent MOSblue cells
From this data (Table 4.4), the transformation efficiencyof the competentMOSBlue cells can be assessed. The number.oftransformants per ug of DNA is the universal.unit used for transformation efficiency. Considering that 0.2ng DNA was used to transform 100 ul cells, plating 5 /!l of this reaction, would be plating 0.01 ng. As seen in Table 4, the amount of colonies on the control transformation 8',was 390.
Table 4.4. The number and phenotype of pMOSBlue transformed E. coli colonies for test and controlligations.
DNA source: Ligation reactions (1-6) pMOSBlue no DNA
Transformation number: 1 2 3 4 5 6 7 8 9
Volume spread (ul) 50 50 50 50 50 50 50 5 40
No. colonies (cfu) . 47 45 42 53 23 0 >500 390 0
No. (~-gancolonies 4 7 8 21 0 0 0 0 0
The transformation therefore produced 390 colonies per 0.01 ng DNA,or 3.9 x 107 colony forming units (cfu) per ug DNA. This is comparable to the expected transformation efficiency forMOSblueof 4 x 107 cfu/ug test plasmid.
The PK reaction, Ill.which the cleaned PCR product was blunt ended (Klenow) and phosphorylated (T4 polynucleotide kinase), was evaluated using the control insert DNA ligated after PK treatment, and no PK treatment. Transformation 4 (Table 4) is the ligation reaction in which the controls insert DNA was PK treated, and transformation 5 the control insert was not treated. As can be seen there are a number of white (recombinant) colonies seen in no. 4, as a result of successful blunt-ending and phosphorylating of DNA termini, and successfulligation with vector DNA. Transformation 5,contains less white colonies,as a
result of the inability to ligate the insert with the vector, as the insert was not treated. This shows that the PK reaction is efficient in preparing the DNA for cloning into pMOSBlue. It also shows that the 23 colonies seen is the approximate amount of background colonies expected from these experiments.This also serves as a control for the ligation reaction.
There are no white colonies seen in transformation 6(Table 5.2), as no insert was included in the ligation. This ensures that white colonies do not appear due to factors other than ligation such as contamination. The appearance of white colonies in the test and control ligations are. entirely due to the.addition of insert DNA to the ligationreactions, A mock transformation (transformation 9) was performed ill which no DNA was transformed, to check for contaminants in the competent cells that may form colonies and false positives. The competent cells shouldn't form colonies, as they do not contain any plasmid DNA conferring ampicillin resistance ..