04/09/13

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Revision as of 12:34, 6 September 2013

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Transformation results

 From the above results,colonies were found growing on the resistance control plate, which indicates that our competent cells already express ampicillin resistance, but this is not meant to be so.

Hence in order to confirm that our results are reliable;
colonies would be randomly selected from each of the two plates (20ul and 200ul) for the 3 TOD genes and
cultured in luria broth + Amp. This broth would be incubated overnight in a 37 degrees shaker incubator.

Gel Electrophoresis

The double digest (pSB1C3) from yesterday was run on a 1% gel.
This was done in order to confirm that the digest worked and also to purify the pSB1C3 vector.

From left to right

Sample 1.1 - track 2
Sample 1.2 - track 3
Sample 2.1 - track 4
Sample 2.2 - track 5

- The 2 Kb bands are the pSB1C3 backbone
- The 1 Kb bands are the RFP biobrick

The two higher bands in tracks 2 and 3 are plasmids that were not digested.

Gel purification of the pSB1C3 backbone and RFP biobrick

The Zymoclean Gel DNA recovery Kit was used to perform the purification and its protocol was followed.
However the elution step was changed to 12ul of elution buffer, and this step was repeated.

Nanodrop of the samples

Sample	Volume	Concentration ng/ul	260/280	260/230
pSB1C3 1	19.8	31.9	1.83	1.75
pSB1C3 2	20	34	1.67	0.92
RFP 1	20	28.8	1.57	0.71
RFP 2	18.8	22.2	1.79	1.84

Double digest of TOD genes PCR products

Samples A (TodX), C (TodF) and E (ToBG) were digested with the enzymes XbaI and SpeI.

Protocol

Samples	A	C	E
Volume (ul)	20	23.5	21
SpeI (ul)	1	1	1
XbaI	0.5	0.5	0.5
Cutsmart Buffer	6	6	6
5mTris Hcl (ul)	32.5	29	31.5

- The samples were incubated at 37C for 90 minutes
- Heat kill the enzymes by incubating the samples at 80C for 20 minutes

Ligation of the TOD genes (X, F and ToBG) to the pSB1C3 backbone

The pGEM-T Vector System was used, However changes were made to the protocol. The promega biomath caculator (www.promega.com/biomath) was used to work out the ratio of insert to vector. The DNA concentration of the TOD genes can be found at Lab work 28/09/2013. The DNA concentration of the pSB1C3 and the RFP biobrick can be found above. The amount of vector DNA was added at 50ng.

Protocol

Samples	A (TodX)	C (TodF)	E (ToBG)	RFP-sample 2 (positive control)	Background control
2X Rapid ligation buffer (ul)	5	5	5	5	5
vector (pSB1C3-sample1) (ul)	1.6	1.6	1.6	1.6	1.6
T4 DNA ligase (ul)	1	1	1	1	1
PCR product (ul)	2.3	1.4	3.1	3.6	-
Deionized water for the final volume of 12 ul	2.1	3	1.3	0.8	4.4

The reaction were incubated overnight at 4C for the maximum number of transformants

@@ Line 14: / Line 14: @@
 ==Transformation results==
-   From the above results,colonies were found growing on the resistance control plate, which indicates that our competent cells already express ampicillin resistance, but this is not meant to be so. Hence in order to confirm that our results are reliable, colonies would be randomly selected from each of the two plates (20ul and 200ul) for the 3 TOD genes and cultured in luria broth + Amp. This broth would be incubated overnight in a 37 degrees shaker incubator.
+   From the above results,colonies were found growing on the resistance control plate, which indicates that our competent cells already express ampicillin resistance, but this is not meant to be so.
+*Hence in order to confirm that our results are reliable;
+*colonies would be randomly selected from each of the two plates (20ul and 200ul) for the 3 TOD genes and
+* cultured in luria broth + Amp. This broth would be incubated overnight in a 37 degrees shaker incubator.
 ==Gel Electrophoresis==
 *The double digest (pSB1C3) from yesterday was run on a 1% gel.