Latest revision as of 22:28, 27 October 2010

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Plasmid Miniprep Experiment

Aims

The aim of this experiment is to extract pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] from E. coli DH5α cells using the Qiagen miniprep kit. The extracted DNA is then analysed using the Nanodrop machine.

Materials and Protocol

Please refer to: Minipreps for Qiagen miniprep protocol, Nanodrop Spectrophotometer for nanodrop protocol and Restriction digests for restriction digestion protocol.

Result

pSB1C3 (No. 1)	pSB1C3 (No. 2)	pSB1C3 (No. 3)	pSB1C3 (No. 4)	lacI (No. 1)	lacI (No. 2)	Double terminator (No. 1)	Double terminator (No. 2)
44.0 µl/ml	19.9 µl/ml	25.0 µl/ml	30.8 µl/ml	10.0 µl/ml	44.2 µl/ml	9.2 µl/ml	39.7 µl/ml

Table 1: Nanodrop spectrophotometer results. The table represents the amount of plasmid present in µl/ml quantity.

Discussion

In our experiment, the concentration of our plasmid DNA range from 19 µg/ml to 44 µg/ml. This value is lower than the expected value of approximately 150 µg/ml for plasmid DNA extraction. The 260/280 nm ratio for all the samples is in the range of 2.0 to 2.4. This indicates that our sample are of low purity.

Conclusion

We are able to obtain plasmid DNA, however the concentartion and the purity of the samples are low. This could be due to the following reasons:

The buffers might have been contaminated
RNAse enzyme might have degraded over time

Solution for the problem

If the buffers of the Qiagen miniprep kit is contaminated, then we will be using the Promega miniprep kit.
If the RNAse enzyme was degraded, then we will be adding 10 µl of fresh RNAse into the P1 buffer.

@@ Line 4: / Line 4: @@
 ==Aims==
-The aim of this experiment is to extract plasmid DNA from pSB1C3 and pSB1AK3 from ''E. coli'' DH5α cells using the Qiagen miniprep kit. The extracted DNA is then analysed using the Nanodrop machine.
+The aim of this experiment is to extract pSB1C3 and [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] with [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] from ''E. coli'' DH5α cells using the Qiagen miniprep kit. The extracted DNA is then analysed using the Nanodrop machine.
 ==Materials and Protocol==
@@ Line 38: / Line 38: @@
 |39.7 µl/ml
 |}
-'''Table 1''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
+'''Table 1''': Nanodrop spectrophotometer results. The table represents the amount of plasmid present in µl/ml quantity.
 ==Discussion==
-We found bands in the lane 2, 3, 4, 5 and 6 showing the presence of plasmid in ''E. coli'' DH5α cells. The ideal concentration of DNA calculated using nanodrop experiment is 150 µg/ml but in the table 1, where all the values have been less than 150 µg/ml which shows that even though there is plasmid present in the cells but it is present in very low amount. Also while doing nanodrop experiment, we measured 260/280 nm ratio for all the samples came out to be between 2.0 to 2.4 approximately which shows that there is a high RNA contamination in the samples.
+In our experiment, the concentration of our plasmid DNA range from 19 µg/ml to 44 µg/ml. This value is lower than the expected value of approximately 150 µg/ml for plasmid DNA extraction. The 260/280 nm ratio for all the samples is in the range of 2.0 to 2.4. This indicates that our sample are of low purity.
 ==Conclusion==
-This experiment shows that there is plasmid present in the ''E. coli'' DH5α cells but they are present in a very low amount and having high RNA contamination possibly due to the following reasons:
+We are able to obtain plasmid DNA, however the concentartion and the purity of the samples are low. This could be due to the following reasons:
-# P1 buffer which contains RNAse might be contaminated.
+# The buffers might have been contaminated
-# RNAse enzyme might have gotten inactive.
+# RNAse enzyme might have degraded over time
 ==Solution for the problem==
-# If P1 buffer of the Qiagen miniprep kit is contaminated, then use a different kit. We have Promega miniprep kit which will be used tomorrow.
+# If the buffers of the Qiagen miniprep kit is contaminated, then we will be using the  Promega miniprep kit.
-# If RNAse enzyme is inactive, then add extra RNAse into the P1 buffer. We would be adding 10 µl in the P1 buffer solution.
+# If the RNAse enzyme was degraded, then we will be adding 10 µl of fresh RNAse into the P1 buffer.
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Team:Newcastle/2 August 2010

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