As such, E. Coli cells that have taken up this plasmid DNA will be resistant to inclining and survive, hence growth of colonies will be observed on the agar plates. One of the rationales behind heat shock method is to create pores, allowing uptake of plasmid DNA (Panda et al. , 2008). For this practical, another ice incubation step is added in after heat shock at ICC to investigate its significance in increasing the efficiency of transformation. It is hypothesized that the additional ice incubation step after heat shock will cause more DNA plasmids uptake by competent E. Lie cells and thus more growth of colonies of successful transforms, resulting in the increase of transformation efficiency. By comparing the results of both standard and mutated protocols, the hypothesis on the effect of post heat shock ice incubation step on transformation efficiency can then be known to be true or false. Materials 1. E. Coli (PUC) culture 2. Eugenia Spin Column with glass fiber matrix 3. Microelectronic tubes 4. Buffer PDP (50 ram Trip-HCI pH 8. 0; 10 mm EDIT; 10 pig/ml Ranges) 5. Buffer PDP (200 mm Noah; 1% SD (w/v)) 6. Buffer PDP (guanidine hydrochloride and acetic acid) .
WI Buffer (guanidine hydrochloride and sopranos) 8. Wash Buffer (70% ethanol) 9. Elution Buffer (10 mm Trip-HCI, pH. 5 at ICC) 10. 1 ml of competent E. Coli cells in mm Cacao 11. Plasmid DNA (FIDDLED) 12. Double distilled (ad) sterile water 13. LB broth 14. LB agar plates 15. LB + inclining agar plates Methods A) Extraction of plasmid DNA: 1. Centrifuge 1. Ml E. Coli cells for 1 minute and discard supernatant. 2. Re- suspend pellet E. Coli cells in pill Buffer PDP. 3. Add pill Buffer PDP and mix by inverting the tube 10 times. Let stand for 2 minutes at room temperature. Add pill Buffer PDP and mix. Centrifuge for 3 minutes 5. Add supernatant to a spin column and centrifuge for 30 seconds. 6. Wash spin column with IPPP WI Buffer and centrifuge for 30 seconds, by pill Wash Buffer and centrifuge again. Discard flow- each round of centrifuge. Followed through after 7. Elute DNA from the spin column with Pl Elution Buffer and centrifuge for 2 minutes. B) Bacterial transformation: 1 . Prepare 3 microfilm tubes, each with 300111 competent cells. 2. Add Pl and Pl extracted plasmid DNA to two tubes. For the third tube (negative intro), add Pl ad water. . Leave on ice for 30 minutes. 4. Heat shock at ICC for 90 seconds. 5. Leave on ice for 10 minutes. (Mutated protocol) 6. Add Mil LB broth and incubate at ICC for 20 minutes. 7. Centrifuge and discard supernatant. Re-suspend in pill LB broth. 8. For the transformed cells, make a 10”1 dilution. 9. Spread pill of neat and 10-1 suspensions on LB+inclining plates. For negative control, spread on LB and LB+inclining plates. 10. Incubate at ICC overnight. 11. Record the relative number of colonies of transforms and calculate transformation efficiency.
Results Transformation efficiency (cuff/Eng) is calculated as follows: Standard protocol AWAY/AWAY = 1. 9 Concentration of plasmid DNA = 139 Eng/Pl Neat suspension: a) Transformation efficiency = 310/ (139 x 5) cuff/Eng b) Transformation efficiency = 401/ (139 x 15) = 0. 192 cuff/Eng 10-1 suspension: a) Transformation efficiency = [8/ (139 x 5)] x 10 0. 115 cuff,’Eng b) Transformation efficiency = [12/ (139 x 15)] x 10 = 0. 0576 cuff/Eng Mutated protocol Concentration of plasmid DNA = 133. 8 Eng/Pl = 0. 446 Calculations of transformation efficiency for both neat and 10-1 suspensions are animal to that of standard protocol.
Table 1 No. Of colonies of successful transforms and transformation efficiencies for different experiments Legend: TM +++ – Too many to count (more than 1000 colonies) AN. – Not applicable Cuff – Colony forming units For both standard and mutated protocols, the ratio of absorbency at Mann and Mann is 1. 9. From Table 1, both the number of colonies and transformation efficiencies for mutated protocol with the additional post heat shock ice incubation step are higher than that of the standard protocol. For Pl of plasmid DNA the neat and 10-1 suspensions of mutated protocol have 456 and 39 colonies respectively.
These are more than that of the standard protocol of 310 and 8 colonies for neat and 10-1 suspensions respectively. For Pl of plasmid DNA the same trend is observed. Mutated protocol has 652 colonies for neat suspension and 52 colonies for 10-1 suspension, which are much higher as compared to the neat and 10-1 suspensions of 401 and 12 colonies respectively for standard protocol. For the negative control which has no plasmid DNA, growth of many colonies (>OHIO) are observed on LB agar plate (in fact, a lawn f bacterial) but no growth is observed on LB+inclining agar plate.
From calculations shown above, standard protocol’s transformation efficiency is the highest for neat suspension of Pl plasmid DNA (0. 446 cuff/Eng), and the lowest for 10-1 suspension of Pl plasmid DNA (0. 0576 cuff/Eng). This same trend is observed for mutated protocol as well (0. 682 cuff/Eng – Pl DNA neat suspension and 0. 259 cuff/Eng- Pl DNA 10-1 suspension). Pl DNA suspensions generally have higher transformation efficiencies than Pl DNA suspensions for both protocols, be it neat or diluted. Discussion The ratio of absorbency at Mann and Mann measured by the spectrophotometers machine is used to determine the purity of DNA and RNA.
A ratio of about 1. 8 implies pure DNA while a ratio of about 2. 0 implies pure RNA (Willingly et al. , 1997). For both protocols, the ratio of 1. 9 means that plasmid DNA that is extracted is not purely DNA and there might be contaminants. The higher number of colonies and transformation efficiencies for mutated protocol as compared to standard protocol shows that the additional ice incubation step after heat shock does have an effect on transformation efficiency. For each type of suspensions, there is an increase of about 1. 5-1. 6 fold of colonies when E. Oil cells are incubated at CO after heat shock. This can be supported by a research study which concluded that incubation of cells on ice after heat shock increases transformation by approximately 1. 6 fold (Sings et al. , 2010). Firstly, during heat shock, there is an increase in thermal motion of plasmid DNA in the competent E. Coli mixture. It is most likely that incubating the suspension on ice after heat shock could decrease this motion which in turn helps to bind more plasmid DNA that are not previously taken up) to the surface of E. Coli (Sings et al. 2010). As such, there would be more successful transforms as more plasmid DNA are further taken up by E. Coli when followed by incubation at 37 Co. Secondly, the purpose of having a post heat shock ice incubation step has an effect on the cell membrane of E. Coli, affecting uptake of plasmid DNA. Lipids from the outer membrane are released during heat shock which rigidify membrane and forms pores on cell surface while cold shock (from ICC to CO) causes fluoridation and disappearance of pores when membrane proteins are released (Panda t al. , 2008).
Thus, incubation at CO after heat shock serves as a second heat shock when the temperature increases from CO to ICC, thereby increasing transformation efficiency as pores are formed again for more DNA uptake. Lastly, the additional ice incubation step allows for the plasmid DNA to convert into Dense resistant form (Bergmann et al. , 1981). This form is essential so that plasmid DNA would not be degraded and the inclining-resistant gene on it can be expressed, ensuring the cells’ survival on LB+inclining agar plate and enhancing transformation.
For negative control, a lawn of E. Coli growth on LB plate and no growth of colonies on LB+inclining plate shows that transformation of competent cells with plasmid DNA is essential for survival of E. Coli in the presence of inclining and the possibility of contamination is eliminated. However, even though the colonies for Pl DNA suspensions are higher than that of Pl DNA suspensions, transformation efficiencies are lower. This could possibly be due to excess DNA. The inverse relationship is in fact established by the above equation for calculating transformation efficiency.