Melbourne/Lab Notebook Weeks 9-12

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Latest revision as of 18:22, 14 October 2007

Week 9

19 Aug 2007

Picked 3-4 transformants from "ligation" plates (17/8) and Grew up liquid cultures.
Transformed the two ligation reactions in the pink rack in the cold room and plated on Gen + Amp plate.

20 Aug 2007

Miniprepped liquid cultures from 19/8 labeled LA, LB, LC and P2 13K A, P2 13K B.
Concentration of DNA was measured to be:
A- 221ng/ul
B-224ng/ul.

Digested:
1. LA X/S
2. LB X/S
3. LC X/S
4. P2 13K A X/S
5. P2 13K B X/S

Desired products:

1. 450bp (95bp different from CI-2 used as control)
2. 450bp
3. 450bp
4. 1kb
5. 1kb

These were run on a gel.

Digestions were set up for ligations.

OmpR-inverter
-Vector: OmpR (AmpR) miniprepped P1 15P from 10/7 digested with S/P.
-Insert: c1 inverter (1kb fragment) P2 13K A with X/P.

RBS-LacZ
-Vector: LacZ (KanR) P2 9E from 6/8 with E/X.
-Insert: RBS (GenR) P4 8J from 6/7 with E/S.

Digested for 2h and heat inactivated for 10min at 80degC.

Made Glycerol Stocks of P2 13K using dry ice method.

21 Aug 2007

Picked 4 colonies from fresh ligation/transformation to Growing up.

- These transformations were made as follows:

- Ligated
  1. 5ul of Purified insert from 17/8 (CI-2 E/S)
  2. 5ul of Redigested P1 5G with E/X and heat inactivated at 60degC for 10min.

Recieved M30109 and CP919.

22 Aug 2007

Miniprepped L1-L4 (liquid cultures from 21/8).
Digested these for ligations.
1. L1 X/P
2. L2 X/P
3. L3 X/P
4. L4 X/P
5. CI-2 X/P (control for 350bp)
6. P1 15P S/P

The above were Ran on a Gel together with P2 13K X/P digest from 21/8.

All L1-L4 had the 450bp desired band.

Gel purified 450bp band from L3 and a 950bp band from the P2 13K digest.

Ligated the following:

OmpR-L3 (RBS-LacZa-double term)
-Vector= P1 15P S/P (above)
-Insert= L3 X/P (above)

OmpR-c1 inverter (P2 13K)
-Vector= P1 15P S/P (above)
-Insert= P1 15P S/P (above)

Ligations were kept overnight at 4degC.

23 Aug 2007

Transformed ligations from 22/8 into competent NM522 cells.

24 Aug 2007

Liquid cultures were made of transformations from 23/8. 6 colonies were picked from each ligation.

25 Aug 2007

Miniprepped cultures from 23/8 and M30109 cells.
Digested:
- 12 minipreps from ligations X/P
- 2 minipreps from M30109 cells S/P
- 2 minipreps from M30109 cells X/P

Ran on a Gel 16 samples that were miniprepped.
Gel purified 1000bp fragment from ompR-c1 inverter ligation digest and a 600bp fragment from ompR-L3 ligation digest.

Week 10

26 Aug 2007

Ligated
1. M30109 S/P to ompR-L3 X/P
2. M30109 S/P to ompR-c1 inv X/P

(all from 25/8) Left overnight at 4degC.

27 Aug 2007

Transformed
1. M30109 S/P to ompR-L3 X/P (26/8)
2. M30109 S/P to ompR-c1 inv X/P (26/8)
3. P1 15P-L3 (from 22/8)
4. P1 15P-c1 inverter (from 22/8)

Plated on Amp plates.

28 Aug 2007

Picked six colonies from each plate (27/8) and cultured at 37degC for 13 hours.

29 Aug 2007

Miniprepped 24 cultures from 29/8.
Diagnosted all with X/S.
Ran on a Gel

-The M30109 ligations yielded random fragments: something wrong with the part? - P1 15P-L3 and P1 15P-c1 inverter ligations had bands of the desired size.

30 Aug 2007

Set up minipreps from 29/8 for sequencing.

OmpR-L3 and OmpR-c1 inverter were the desired sequences.

Parts created:

OmpR-RBS-LacZa-double terminator

OmpR-c1 inverter

31 Aug 2007

1 Sept 2007

Week 11

2 Sept 2007

Digested and Ran on a Gel M30109 minipreps from 25/8 using a number of restriction enzymes to try to find out what it is:

1. EcoRV
2. Xho1
3. EcoRV/Xho1
4. E/P
5. E/S
6. E/X
7. X/P
8. X/S
9. S/P
10. E
11. P
12. S
13. EcoR1/EcoRV/Xho1
14. Apa1
15. BamH1
16. EcoR1/BamH1

The results showed that although a fragment of cph8 may be present in the part (1200bp fragment present only when double digested by EcoRV/Xho1), it may not be in the form as designated in the iGEM wiki.

The original miniprep was sent off for sequencing using vf2. The results showed that the part is a form of RBS-cph8, although the sequencing got cut off in the middle of the cp8 sequence. Therefore, the entire part designated is not present. We consequently decided to create the M30109 part ourselves.

3 Sept 2007

4 Sept 2007

Made LB (600ml) + LB-agarose (400ml)
Created Agar Plates with Kan.

5 Sept 2007

Recieved ComP and ComA sequences from GeneArt.

6 Sept 2007

Transformed the following on Amp plates except where labeled
1. ComA
2. ComP (Kan plate)
3. 6kb band from undigested M30109
4. 10kb band from undigested M30109
5. RBS-LacZa-double terminator from 22/8
6. OmpR-RBS-LacZa-double terminator from 29/8
7. OmpR-RBS-LacZa-double terminator from 29/8 into CP919 EnvZ- strains.

7 Sept 2007

Liquid cultured a colony from each of the 6/9 transformations. No growth was observed for the M30109 undigested plates and were discarded.

8 Sept 2007

Miniprepped cultures from 7/9.
Digested these with X/P
Ran these on a Gel.

-All had desired sizes.

Glycerol Stocks of all four of these were created.

Week 12

9 Sept 2007

Ligated the following:

ComP-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComP X/P fragment from 8/9.

ComA-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComA X/P fragment from 8/9.

10 Sept 2007

11 Sept 2007

12 Sept 2007

Transformed ligations from 9/9.
Also Transformed resuspended DNA for the following:
1. [http://partsregistry.org/Part:BBa_I15008 P2 21A] (ho1)
2. [http://partsregistry.org/Part:BBa_I15009 P2 21A] (PcyA)
3. [http://partsregistry.org/Part:BBa_J61034 P4 8J] (IPTG sensory device)
4. [http://partsregistry.org/Part:BBa_R0040 P1 7O] (tetR negative promoter)
5. [http://partsregistry.org/Part:BBa_B0015 P1 1I] (Double terminator)

and minipreprs from previous dates:

1. P4 8J from 5/7
2. P1 5G

Efficiency of fast ligations were simultaneously tested for. Using 2X ligase buffer, 2hour and 5min ligations were tested using ComA parts from 8/9. These did not have ligation efficiencies as high as the overnight ligations but were sufficient. The 5 min ligation seemed to have more colonies than the 2 hour ligation.

13 Sept 2007

P4 8J and P1 5G were overgrown so were streaked onto new plates.
Colony PCR using The Endy Procol [http://openwetware.org/wiki/Endy:Colony_PCR] of 2 colonies from ComA-cell death (12/9) and OmpR-L3 (27/8),

1. 5ul 10 X PCR buffer (green)
2. 5ul MgCl2 25mM
3. 0.5ul vf2
4. 0.5ul vR
5. 1.0ul dNTPs 25mM
6. 0.5ul GoTaq
7. 37.5ul milliQ water

on a cycle of:

1. 94C for 5min
2. 94C for 30 seconds
3. 55C for 30 seconds
4. 72C for 1:15 minutes
5. repeat 2-4 for 30 times
6. 72C for 7min.

14 Sept 2007

15 Sept 2007

@@ Line 177: / Line 177: @@
 OmpR-L3 and OmpR-c1 inverter were the desired sequences.
-<BR>
+<BR><BR>
 <font color=green size=3>
 Parts created: <BR><BR>
 [[Melbourne/OmpR-RBS-LacZa-double terminator|OmpR-RBS-LacZa-double terminator]]
-[[Melbourne/OmpR-c1 inverter|P115-P213K]]
+[[Melbourne/OmpR-c1 inverter|OmpR-c1 inverter]]
 </font>
@@ Line 199: / Line 199: @@
 </b>
 </font><BR>
+*[[Melbourne/Diagnostic Digest| Digested]] and [[Melbourne/Loading a DNA gel|Ran on a Gel]] [[Melbourne/M30109| M30109]] minipreps from 25/8 using a number of restriction enzymes to try to find out what it is:
+*# EcoRV
+*# Xho1
+*# EcoRV/Xho1
+*# E/P
+*# E/S
+*# E/X
+*# X/P
+*# X/S
+*# S/P
+*# E
+*# P
+*# S
+*# EcoR1/EcoRV/Xho1
+*# Apa1
+*# BamH1
+*# EcoR1/BamH1
+The results showed that although a fragment of cph8 may be present in the part (1200bp fragment present only when double digested by EcoRV/Xho1), it may not be in the form as designated in the iGEM wiki.
+The original miniprep was sent off for sequencing using [[Melbourne/primary vf2|vf2]]. The results showed that the part is a form of RBS-cph8, although the sequencing got cut off in the middle of the cp8 sequence. Therefore, the entire part designated is not present. We consequently decided to create the [[Melbourne/M30109| M30109]] part ourselves.
@@ Line 209: / Line 233: @@
 </b>
 </font><BR>
+*Made [[Melbourne/Secondary Reagent LB|LB]] (600ml) + [[Melbourne/Secondary Reagent LB|LB]]-agarose (400ml)
+*Created [[Melbourne/Secondary Reagent Agar Plates|Agar Plates]] with Kan.
@@ Line 214: / Line 241: @@
 </b>
 </font><BR>
+*Recieved ComP and ComA sequences from GeneArt.
@@ Line 220: / Line 249: @@
 </font><BR>
+*[[Melbourne/Transformation Protocol|Transformed]] the following on Amp plates except where labeled
+*# ComA
+*# ComP (Kan plate)
+*# 6kb band from undigested [[Melbourne/M30109| M30109]]
+*# 10kb band from undigested [[Melbourne/M30109| M30109]]
+*# RBS-LacZa-double terminator from 22/8
+*# OmpR-RBS-LacZa-double terminator from 29/8
+*# OmpR-RBS-LacZa-double terminator from 29/8 into [[Melbourne/CP919| CP919]] EnvZ- strains.
 <font size=3><b>7 Sept 2007
 </b>
 </font><BR>
+*[[Melbourne/Growing up cells|Liquid cultured]] a colony from each of the 6/9 transformations. No growth was observed for the [[Melbourne/M30109| M30109]] undigested plates and were discarded.
@@ Line 230: / Line 269: @@
 </font><BR>
+*[[Melbourne/Miniprep protocol|Miniprepped]] cultures from 7/9.
+*[[Melbourne/Diagnostic Digest| Digested]] these with X/P
+*[[Melbourne/Loading a DNA gel|Ran these on a Gel]].
+-All had desired sizes.
+*[[Melbourne/Making glycerol Stocksl|Glycerol Stocks]] of all four of these were created.
 ==Week 12==
@@ Line 236: / Line 281: @@
 </b>
 </font><BR>
+*[[Melbourne/Ligation Protocol|Ligated]] the following:
+<font color=crimson> ComP-backbone vector </font><BR>
+-Vector: [[Melbourne/BBa_P1010_A|P3 20I]] Death Plasmid AmpR X/P fragment from 8/9. <BR>
+-Insert: ComP X/P fragment from 8/9.
+<font color=crimson> ComA-backbone vector </font><BR>
+-Vector: [[Melbourne/BBa_P1010_A|P3 20I]] Death Plasmid AmpR X/P fragment from 8/9.<BR>
+-Insert: ComA X/P fragment from 8/9.
@@ Line 251: / Line 307: @@
 </b>
 </font><BR>
+*[[Melbourne/Transformation Protocol|Transformed]] ligations from 9/9.
+*Also [[Melbourne/Transformation Protocol|Transformed]] resuspended DNA for the following:
+*# [http://partsregistry.org/Part:BBa_I15008 P2 21A] (ho1)
+*# [http://partsregistry.org/Part:BBa_I15009 P2 21A] (PcyA)
+*# [http://partsregistry.org/Part:BBa_J61034 P4 8J] (IPTG sensory device)
+*# [http://partsregistry.org/Part:BBa_R0040 P1 7O] (tetR negative promoter)
+*# [http://partsregistry.org/Part:BBa_B0015 P1 1I] (Double terminator)
+and minipreprs from previous dates:
+*# [[Melbourne/BBa_J61035|P4 8J]] from 5/7
+*# [[Melbourne/BBa_C0051|P1 5G]]
+* Efficiency of fast ligations were simultaneously tested for. Using [[Melbourne/primary 2X Ligase buffer|2X ligase buffer]], 2hour and 5min ligations were tested using ComA parts from 8/9. These did not have ligation efficiencies as high as the overnight ligations but were sufficient. The 5 min ligation seemed to have more colonies than the 2 hour ligation.
@@ Line 257: / Line 327: @@
 </font><BR>
+*[[Melbourne/BBa_J61035|P4 8J]] and [[Melbourne/BBa_C0051|P1 5G]] were overgrown so were streaked onto new plates.
+*[[Melbourne/Colony PCRl|Colony PCR]] using The Endy Procol [http://openwetware.org/wiki/Endy:Colony_PCR] of 2 colonies from ComA-cell death (12/9) and OmpR-L3 (27/8),
+*# 5ul 10 X PCR buffer (green)
+*# 5ul MgCl2 25mM
+*# 0.5ul [[Melbourne/primary vf2|vf2]]
+*# 0.5ul [[Melbourne/primary vR|vR]]
+*# 1.0ul dNTPs 25mM
+*# 0.5ul GoTaq
+*# 37.5ul [[Melbourne/primary milliq|milliQ water]]
+on a cycle of:
+*# 94C for 5min
+*# 94C for 30 seconds
+*# 55C for 30 seconds
+*# 72C for 1:15 minutes
+*# repeat 2-4 for 30 times
+*# 72C for 7min.
 <font size=3><b>14 Sept 2007

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