Revision as of 03:14, 26 October 2010

Miniprep for pGFPrrnB with yneA

The aim of this experiment is to produce more stocks of yneA in pGFPrrnB.

Tube 1	Tube 2	Tube 3	Tube 4	Tube 5	Tube 6	Tube 7	Tube 8	Tube 9	Tube 10	Tube 11	Tube 12
286.1 µl/ml	304.0 µl/ml	316.3 µl/ml	421.6 µl/ml	518.7 µl/ml	460.1 µl/ml	370.1 µl/ml	377.3 µl/ml	346.0 µl/ml	347.4 µl/ml	202.8 µl/ml	307.4 µl/ml

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

The concentration ranged from 286.1 µl/ml to 518.7 µl/ml. Therefore we have obtained high concentration of yneA in pGFPrrnB.

To linearise the vector pSB1C3. The linear vector will be used as a PCR template for Gibson cloning of the Subtilin immunity and rocF BioBricks.

We are doing a single digest with HinDIII.

Gel electrophoresis results for digestion:

Figure 1: Gel electrophoresis of the amplified PCR products and restriction digest

The digestion was successful — the correct band was seen. We can proceed to gel extraction.

@@ Line 47: / Line 47: @@
 The concentration ranged from 286.1 µl/ml to 518.7 µl/ml. Therefore we have obtained high concentration of ''yneA'' in pGFPrrnB.
-=Single and Double Digestion of pGFPrrnB with ''yneA''=
-==Aims==
-The aim of this experiment is to check if the insert ''yneA'' has been inserted into vector pGFPrrnB.
-==Materials and Protocol==
-We are doing two digests for pGFPrrnB and ''yneA'':
-* Single digest with HinDIII;
-* Double digest with EcoR1 and Nhe1.
-Please refer to [[Team:Newcastle/Restriction_digests|restriction digests]] and [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]].
-==Results==
-Gel electrophoresis results for digestion: