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Problem

Gel extraction and ligation of filamentous cell part into pGFPrrnB and pSB1C3

Aims

The aim of the experiment is to further purify the filamentous cell part (yneA), pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together.

Materials and Protocol

Please refer to gel electrophoresis, gel extraction, nanodrop spectrophotometer and ligation.

Ligation mix:

Reagents	1:3(μl)	1:5(μl)	Vector(μl)
Vector	3	2	1.5
Insert	3	6	7.5
10X BUFFER	1	1	1.1
T4 Ligase	1	1	1
H2O	2	0	0
Total Volume	10.0	10.0	10.0

Table 1: Ligation mix for ligation of yneA into pGFPrrnB and pSB1C3

Results

	yneA 1	yneA 2	yneA 3	pSB1C3	pGFPrrnB
Concentration of DNA ng/µl	3.4	4.7	5.4	2.2	18.5

Table 2: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.

Discussion

The DNA concentration obtained for yneA ranged from 3.4 µl/ml to 5.4 µl/ml, 2.2 µl/ml for pSB1C3 and 18.5 µl/ml for pGFPrrnB . Therefore the concentration for yneA and pSB1C3 is low.

Conclusion

We will proceed with digestion, but another set of ligation will be set up to repeat the process again.

Results and Conclusion

Ligation will be done overnight because concentration of the filamentous cell part, pGFPrrnB and pSB1C3 are not very good. Please refer to 20.08.10 for results and discussion. Overnight culture of yneA, pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010.

@@ Line 5: / Line 5: @@
 ==Aims==
-The aim of the experiment is to further purify the filamentous cell part, pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together.
+The aim of the experiment is to further purify the filamentous cell part (''yneA''), pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together.
 ===Materials and Protocol===
@@ Line 86: / Line 86: @@
 Ligation will be done overnight because concentration of the filamentous cell part, pGFPrrnB and pSB1C3 are not very good. Please refer to [[Team:Newcastle/20_August_2010|20.08.10]] for results and discussion. Overnight culture of ''yneA'', pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010.
-=Rehydration of new subtilin immunity primers and PCR amplification=
-==Aims==
-The aim of the experiment is to rehydrate the new primers, Prom_For, pSB1C3_for, pSB1C3_rev and Term_rev. The primers is then used to amplify the ''spaIFEG'' gene cluster, Promoter and RBS (pVeg-SpoVG) and Double terminator .
-These primers were ordered as a solution to the problem we encountered before we were about to attempt carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. The primers will be used with previously rehydrated primers to try and obtain all four parts that we need to make the Subtilin Immunity BioBrick using the Gibson Protocol. So far the ''spaIFEG'' coding sequence (part 3) has already been successfully obtained. We still need to obtain the plasmid vector (part 1), promoter & RBS (part 2) and double terminator (part 4). So in order to do this the primers received today will be rehydrated and Phusion PCR will be carried out for each of the parts in order to amplify these parts.
-==Materials and protocol==
-Please refer to  [[Team:Newcastle/DNA_re-hydration| DNA rehydration]] page for the protocol for the rehydration of the four primers and to the [[Team:Newcastle/PCR| Phusion PCR]] page for protocol followed for Phusion PCR.
-{|border=1
-|-
-!'''Tube'''
-!'''Part to be amplified'''
-!'''DNA fragment consisting the part'''
-!'''Forward primer'''
-!'''Reverse Primer'''
-!'''Melting Temperature (Tm in °C) '''
-!'''Size of the fragment (in bp)'''
-!'''Extension time* (in seconds)'''
-|-
-|1
-|Plasmid Vector
-|pSB1C3
-|'''pSB1C3_for'''
-|'''pSB1C3_rev'''
-|68
-|2046 +
-|70
-|-
-|2
-|Promoter and RBS (pVeg-SpoVG)
-|BioBrick Bba_K143053
-|'''Prom_for'''
-|P2P2_rev
-|67
-|139 +
-|15
-|-
-|4
-|Double terminator
-|pSB1AK3 consisting BBa_B0014
-|P1T1_for
-|'''Term_rev'''
-|63
-|116 +
-|15
-|}
-'''Table 1''': This table shows the three different Phusion PCR reactions that were carried out today. If this is successful, these three parts (part 1, 2 and 4) along with (part 3) which is already obtained can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
-*The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
-*The primers in bold are the '''new primers''' we received today. The primers not in bold were previously re-hydrated.
-== Discussion and Conclusion ==
-Gel electrophoresis will be carried out tomorrow, 20th August, 2010 to check for the amplified ''spaIFEG'' gene cluster, Promoter and RBS (pVeg-SpoVG) and Double terminator.
-'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
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Team:Newcastle/19 August 2010

From 2010.igem.org