Transformation


 * Home ==> Tranformation**

//Edited by Nick Pagani//
 * Welcome to the Tranformation Page!**

There are essentially two transformations which must take place throughout the course of this experiment. First, competent DH5a cells must be transformed with L4440 plasmids cloned with the DPY-10 insert. This will allow us to produce much more of the DNA we desire, the L4440 plasmids with the DPY-10 insert, as the cells reproduce. Performing a miniprep on a transformed colony will allow us to isolate the DNA of interest. The second transformation which must occur involves the HT115, or feeding strain //E.coli// cells. These cells must be transformed with the L4440 plasmids cloned with the DPY-10 insert after the DH5a cells have produced many copies. This will increase our effectiveness and efficiency of the second transformation. After transformation the HT115 cells can then be fed to our worms in order to cause RNAi.

1. Pre-warm AMP plates at 37 degrees C. 2. Add 5 ul of ligated DNA (L4440+DPY-10) to 100 ul of DH5a competent cells*. 3. Incubate on ice for 60 minutes. 4. Add to 420 ul of LB. 5. Incubate transformation tube at 37 degrees Celsius for 60 minutes. 6. Plate 250 ul of transformants onto Pre-warmed AMP plates. (250 ul onto each plate, should result in 2 plates total)
 * Transformation Procedure**

Before beginning this procedure, add two .25 ml samples of fresh overnight //E.coli// culture which have been grown in LB to two separate tubes containing 25 ml of SOB ** shaking vigorously at 20-25 degrees C. These cultures should be allowed to grow for 10-36 hours. 1. Place cultures on ice for 10 minutes. 2. Pellet cells @ 2500x g for 6 minutes at 0-4 degrees Celsius. 3. Discard supernatant and resuspend cells in 1 ml ice cold 1X Wash Buffer*. 4. Repeat step 1 to repellet cells. 5. Discard supernatant completely and resuspend cells in 1ml 1 X Competent Buffer*. 6. Aliquot 100 ul of competent cells in 1.5 ml sterile microfuge tubes.
 * Preparation of Competent Cells Procedure
 * The Wash and Competent Buffers are 2X concentration at stock and must be diluted to 1X. 1X Wash Buffer: 1 ml 2X Wash Buffer + 1 ml Dilution Buffer. Competent Buffer: 1 ml 2X Competent Buffer + 1 ml Dilution Buffer.

It would be advisable to perform a control transformation in order to verify that the competent cells used were in fact competent and could be transformed. We know that the pUC19 plasmid can be easily inserted into competent cells. Therefore, as a control, repeat the same procedure above, except add 5 ul of the pUC19 plasmid to 100 ul of the DH5a competent cells. These cells should also be plated on Pre-warmed AMP plates. If the cells were in fact competent, growth should be evident on these plates. If the positive control works, but there is no growth is apparent on the plates from the transformation of the ligation, we can conlcude that the source of error was not the competency of the cells, but a problem with the ligation.


 * Results**

__Trial 1__ Unfortunately, we have concluded that our initial transformation was unsuccessful. The colonies we thought to be transformed were grown on plates containing ampicillin. However, the colonies grew with a strange reddish color, leading us to believe that they were in fact not the transformed //E.coli// we desired. Possibilities for this almost unexplainable oddity include contamination, however, this is curious because we plated the cells on ampicillin. Although we recognized the colonies as most likely foreign or unsuccessful we attempted to miniprep six colonies in order to isolate any plasmids which the cells may have contained. We then performed PCR on the miniprep results and ran a gel to observe our product. On the gel a band only appeared in our positive control lane. This is somewhat hopeful as it indicates that our PCR procedure was successful, however, the absence of any bands in the six lanes of the minipreps from our six cultures leads us to the conclusion that our transformation was unsuccessful. Therefore, we have begun to repeat our procedure, beginning with another digestion of the L4440 plasmids and the DPY-10 inserts, a ligation of these, and finally a second attempt at transformation. We have been sure to verify our results along each step of the way with various gels and have proceded much more cautiously and prudently in order to ensure that our results this time around will have much greater success.

The two gels displaying the results of Trial 1 can be seen below. The number 1 and the letters A, B, C, D and E coordinate to the specific cultures we isolated each DNA sample from.The +1 and +2 lanes indicate positive controls and the (-) lane is a negative control.



__Trial 2__

According to our gel results, it appears as if our second attempt at transformation was successful. Although the gel is blurred and the bands hard to make out, one can clearly observe somewhat of a band in the PCR lane. The fact that the miniprep lane has no band simply indicates that little DNA was isolated from the miniprep, as is a possibility. However, the strong presence in the PCR lane shows that what DNA was isolated in the miniprep was amplified substantially, therefore some DNA had to be present from the miniprep. This band in the PCR lane likely correlates to the DPY-10 insert, indicating that the L4440 plasmids were successfully cloned with the insert. Our positive and negative control lanes both appear as they should. Thus, from our gel we can conclude that the transformation worked and that the successfully transformed colonies contain L4440 plasmids with the DPY-10 insert.

Our gel results from Trial 2 can be seen below. MP correlates to the miniprep sample. The PCR lane contains our PCR product and the (+) and (-) lanes are our positive and negative controls, respectively. Below the gel you can see images of our transformed colonies. The control plate contains DH5a cells transformed with just L4440 plasmids. Growth on this plate verifies that the cells were in fact competent and able to accept the plasmids.



Now that we know our second trial at transformation was successful we can continue proceeding through the remaining steps of our procedure. The next step would be to transform competent HT115 cells with the isolated DNA from the miniprep we performed on the transformed DH5a cells. The HT115 strain of //E.coli// is a feeding strain for our worms. The procedure for the preparation of competent HT115 cells is the same as seen above. The transformation procedure should also be followed as above.