=Gel extraction and ligation of filamentous cell part into pGFPrrnB and pSB1C3=
-
==Gel extraction==
+
==Aims==
-
===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.
-
+
-
To purify the products of ''yneA'', pGFPrrnB and PSB1AT3 by extracting the bands from the gel after running gel electrophoresis. Concentration of the DNA and vectors are then checked with nanodrop.
+
===Materials and Protocol===
===Materials and Protocol===
-
Please refer to
+
Please refer to [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]], [[Team:Newcastle/Gel_extraction|gel extraction]], [[TeamNewcastleNanoDrop_Spectrophotometer|nanodrop spectrophotometer]] and [[Team:Newcastle/Ligation|ligation]].
The bands we extracted from the gel was good for pGFPrrnB, but not so strong for ''yneA'' and PSB1C3.
+
-
+
-
The results we got from the nanodrop were not as good as expected for ''yneA'' and PSB1C3.
+
-
+
-
===Conclusion===
+
-
+
-
We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
+
-
+
-
==Ligation==
+
-
+
-
===Aims===
+
-
+
-
To ligate ''yneA'' into both vectors pGFPrrnB and pSB1C3.
+
-
+
-
===Materials and Protocol===
+
-
+
-
Please refer to [[Team:Newcastle/Ligation|ligation]].
+
-
+
-
====Ligation mix for pSB1C3 with ''yneA''====
+
-
+
-
The concentration of pSB1C3 and ''yneA'' that we got from the gel extraction earlier today was very low, so we used a different mixture set up for ligation.
+
Ligation mix:
Ligation mix:
Line 100:
Line 51:
|}
|}
-
===Results and Conclusion===
+
'''Table 1''': Ligation mix for ligation of ''yneA'' into pGFPrrnB and pSB1C3
-
We will leave the ligation overnight because concentration of ''yneA'', pGFPrrnB and pSB1C3 are not very good. Please refer to [[Team:Newcastle/20_August_2010|20.08.10]] for results and discussion.
+
===Results===
-
+
-
==Digestion==
+
-
+
-
===Aim===
+
-
+
-
To repeat digestion from [[Team:Newcastle/18_August_2010|yesterday]].
+
-
+
-
===Materials and Protocol===
+
-
+
-
Please refer to [[Team:Newcastle/Restriction_digests|restriction digest]].
+
-
+
-
+
-
==Setting up Overnight Cultures==
+
-
+
-
===Aims===
+
-
+
-
To prepare cultures for miniprep of ''yneA'', pGFPrrnB and pSB1C3 [[Team:Newcastle/20_August_2010|tomorrow]].
+
-
+
-
===Materials and Protocol===
+
-
+
-
Please refer to [[Team:Newcastle/Growing_an_overnight_cultures|growing an overnight culture]].
+
-
+
-
+
-
+
-
+
-
=Subtilin Immunity=
+
-
+
-
+
-
+
-
==Aims==
+
-
+
-
[[Image:Newcastle 4 Primer Tubes.jpg|150px|right]]
+
-
Today we received the four new primers that we ordered:
+
-
*Prom_For
+
-
*pSB1C3_for
+
-
*pSB1C3_rev
+
-
*Term_rev
+
-
+
-
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. Table # below shows the different fragments, primers and conditions used for each tube.
+
{|border=1
{|border=1
|-
|-
-
!'''Tube'''
+
!
-
!'''Part to be amplified'''
+
!''yneA'' 1
-
!'''DNA fragment consisting the part'''
+
!''yneA'' 2
-
!'''Forward primer'''
+
!''yneA'' 3
-
!'''Reverse Primer'''
+
!pSB1C3
-
!'''Melting Temperature (Tm in °C) '''
+
!pGFPrrnB
-
!'''Size of the fragment (in bp)'''
+
|-
-
!'''Extension time* (in seconds)'''
+
!Concentration of DNA ng/µl
-
|-
+
!3.4
-
|1
+
!4.7
-
|Plasmid Vector
+
!5.4
-
|pSB1C3
+
!2.2
-
|pSB1C3_for
+
!18.5
-
|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.
+
'''Table 2''': Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
-
*The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
+
-
*The primers in bold are the '''new primers''' we received today. The primers not in bold were previously re-hydrated.
+
-
+
-
== Discussion and Conclusion ==
+
-
Tommorrow we will carry out a test gel to check the sizes of the amplified fragments and if the sizes are correct then gel extraction will be performed
+
-
+
-
=''rocF'' BioBrick=
+
-
+
-
==Gel extraction of linearised pSB1C3==
+
-
+
-
===Aims===
+
-
+
-
Following [[Team:Newcastle/19_August_2010| yesterday]]'s Phusion PCR reactions, we plan to first perform gel electrophoresis of the amplified DNA sequences to check if they are of the correct sizes. If they appear to be successful we will then perform gel extraction, and finally nanodrop to record the level of DNA.
+
-
+
-
===Materials and protocol===
+
-
+
-
Please refer to the:
+
-
*[[Team:Newcastle/Gel_electrophoresis| gel electrophoresis]], [[Image:Newcastle Gel 1 (20-08-10).jpg|right|300px|The gel loaded up with the amplified PCR products (blue) and the ladders on either side (green), carried out to check the size of the sequences. Please see results section below for key.]]
+
-
*[[Team:Newcastle/Gel_extraction| gel extraction]] and
[[Image:Newcastle Gel 20-08-2010.jpg|700px|centre]]
+
-
+
-
'''Figure 1''': Gel electrophoresis of the amplified PCR products
+
-
+
-
*'''Lane 1''': 1 Kb ladder
+
-
*'''Lane 2''': pSB1C3 (for Subtilin Immunity)
+
-
*'''Lane 3''': pSB1C3 (for ''rocF'')
+
-
+
-
*'''Lane 4''': pVeg (for Subtilin Immunity)
+
-
*'''Lane 5''': terminator (for Subtilin Immunity)
+
-
*'''Lane 6''': pSpacoid (for ''rocF'')
+
-
*'''Lane 7''': terminator (for ''rocF'')
+
-
*'''Lane 8''': 100 bp ladder
+
===Discussion===
===Discussion===
-
After looking at the results of our gel, there are no distinct band in lanes 2-3, therefore pSB1C3 vector did not work for both Subtilin Immunity or ''rocF''. However, there are single bands in lanes 4-7 at similar positions to each other. They are about ... in size which is what is expected.
+
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.
-
[[Image:Newcastle Gel 2 (20-08-10) - extraction.jpg|right|300px]]
+
===Conclusion===
-
[[Image:Newcastle Gel 3 (20-08-10) - extraction.jpg|300px|right]]
+
-
As lanes 2-3 showed no bands, we decided to make a range of Tms to perform another set of PCR reactions to check whether the Tm was the problem. Three Tms were used: 60°C, 65°C and 70°C and six tubes were set up; a duplicate was created for each Tm. After the PCR reactions were finished, there still wasn't any visible pSBIC3 vector band showing up. The problem could have occurred due to the working stocking solution for our primers. Our next step was to re-make the working stock solution for our primers. Also, we had decided to use the Tms 55°C, 60°C, 65°C, 70°C - duplicate for each, making eight PCR tubes in total.
+
-
As lanes 4-7 had bands at the right positions, gel extraction of these four parts was carried out. The concentration of DNA was then measured using the Nanodrop spectrophotometer.
+
We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
-
===Conclusion===
+
===Results and Conclusion===
-
+
-
The PCR machines were left to run and results of the eight PCR reactions at the 4 different temperatures shall be obtained on [[Team:Newcastle/23_August_2010| Monday]]. After a test gel has been run and if bands appear at the right sizes then gel extraction will be carried out.
+
-
==PCR of pSB1C3, pspac oid and double terminator with new primers==
+
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.
-
'''Go back to our main [[Team:Newcastle/notebook| Lab book]] page'''
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.
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.
"
