Revision as of 14:20, 27 October 2010

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Problem

pH changing for HEPES

We prepared 100mM of HEPES (it is a dibasic compound) at pH 7.0 and sent it for autoclaving. N.B. During autoclaving we loose some volume of the solution.

yneA

Aims

The aim of the experiment is to test if the filamentous cell part was cloned into pGFP-rrnB successfully.

Materials and protocols

Please refer to: Plasmid extraction.
Please refer to:Restriction digest. We used Nhe1 and Spe1 to remove the yneA from pGFP-rrnB.
Please refer to: Gel electrophoresis for running all the digested plasmid fragments.

Results

Figure 1: Gel electrophoresis result for restriction digest of pGFPrrnB and yneA with Nhe1 and Spe1.

Lane 1: 1kb DNA ladder
Lane 2: Tube 1
Lane 3: Tube 2
Lane 4: Tube 3
Lane 5: Tube 4
Lane 6: Tube 5
Lane 7: Tube 6
Lane 8: Tube 7
Lane 9: Tube 8
Lane 10: Tube 9
Lane 11: Tube 10
Lane 12: Tube 11
Lane 13: Tube 12
Lane 14: 1kb DNA ladder

Conclusion

The results show that the digest works, there is a band at approximately 541bp corresponding to yneA and a band at approximately 8.4kbp corresponding to pGFPrrnb in lanes 2, 3, 4, 6, 7, 8, 9, 11 and 12.

@@ Line 5: / Line 5: @@
 N.B. During autoclaving we loose some volume of the solution.
-=Transformation of ''Bacillius subtilis'' 168 with pGFP-rrnB containing filamentous cell part=
+=''yneA''=
-==Aim==
+==Aims==
-The aim of the experiment is to transform ''Bacillus subtilis'' 168 with the plasmid pGFP-rrnB containing our filamentous cell part. After transformation the plasmid will integrate into the chromosome at ''amyE''. This integration removes the ability to break down starch, allowing us to select for our desired construct using the iodine test.
+The aim of the experiment is to test if the filamentous cell part was cloned into pGFP-rrnB successfully.
-[[Image:filamentous_in_pgfprrnb.jpg|800px]]
+==Materials and protocols==
+#Please refer to: [[Team:Newcastle/Qiagen_Minipreps#Plasmid_extraction| Plasmid extraction]].
+#Please refer to:[[Team:Newcastle/Restriction_digests|Restriction digest]]. We used Nhe1 and Spe1 to remove the ''yneA'' from pGFP-rrnB.
+#Please refer to:[[Team:Newcastle/Gel_electrophoresis| Gel electrophoresis]] for running all the digested plasmid fragments.
+==Results==
+[[Image:03.09.10.png#file|400px]]
+'''Figure 1''': Gel electrophoresis result for restriction digest of pGFPrrnB and ''yneA'' with Nhe1 and Spe1.
+* '''Lane 1''': 1kb DNA ladder
+* '''Lane 2''': Tube 1
+* '''Lane 3''': Tube 2
+* '''Lane 4''': Tube 3
+* '''Lane 5''': Tube 4
+* '''Lane 6''': Tube 5
+* '''Lane 7''': Tube 6
+* '''Lane 8''': Tube 7
+* '''Lane 9''': Tube 8
+* '''Lane 10''': Tube 9
+* '''Lane 11''': Tube 10
+* '''Lane 12''': Tube 11
+* '''Lane 13''': Tube 12
+* '''Lane 14''': 1kb DNA ladder
+==Conclusion==
+The results show that the digest works, there is a band at approximately 541bp corresponding to ''yneA'' and a band at approximately 8.4kbp corresponding to pGFPrrnb in lanes 2, 3, 4, 6, 7, 8, 9, 11 and 12.
-==Materials and Protocol==
-Please refer to: [[Team:Newcastle/Transformation of B. subtilis| Transformation of ''Bacillus subtilis'']]
-Note: Overnigth culture of ''B. subtilis'' 168 in MM competence medium was done the day before and the iodine test was performed the day after transformation.
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Team:Newcastle/2 September 2010

From 2010.igem.org