Glasgow/Wetlab/Week7

(Difference between revisions)

Revision as of 14:57, 24 August 2007

Lynsey did colony PCR (see Protocol 9) on the following E. coli TOP10 transformants to check that their plasmids carry our expected inserts:

Plasmid	Expected Insert	Primers
pCR4	Pu	Pu_Prefix_After & Pu_Suffix_After
pCR4	Pu	Pu_Prefix_Emma & Pu_Suffix_Emma
pCR4	Pr	Pr_Prefix_1 & Pr_Suffix_1
pCR4	XylR	XylR_Prefix_1 & XylR_Suffix_1
pCR4	XylR & Pr	Pr_Prefix_1 & XylR_Suffix_1

Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.

Maija did colony PCR (see Protocol 9) on the following E. coli DB3.1 transformants to check that their plasmids carry our expected inserts:

Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.

Maia took the following BioBricks from the kit plates and transformed (see Protocol 2) into E. coli TOP10 cells:

BioBrick Part	Function	Chassis	Plasmid	Kit Plate Location (Plate/Well)
[http://partsregistry.org/Part:BBa_B0014 Bba_B0014]	Double terminator	E. coli TOP10	pSB1AK3	1/1G
[http://partsregistry.org/Part:BBa_B0015 Bba_B0015]	Double terminator	E. coli TOP10	pSB1AK3	1/1I
"	"	"	"	3/3O
[http://partsregistry.org/Part:BBa_J61100 Bba_J61100]	RBS	E. coli TOP10	pSB1A2	4/12N
[http://partsregistry.org/Part:BBa_J61101 Bba_J61101]	RBS	E. coli TOP10	pSB1A2	4/12J
[http://partsregistry.org/Part:BBa_J61102 Bba_J61102]	RBS	E. coli TOP10	pSB1A2	4/12L
[http://partsregistry.org/Part:BBa_J61103 Bba_J61103]	RBS	E. coli TOP10	pSB1A2	4/12P
[http://partsregistry.org/Part:BBa_E0430 Bba_E0430]	EYFP + RBS + terminator	E. coli TOP10	pSB1A2	1/11A
[http://partsregistry.org/Part:BBa_E0422 Bba_E0422]	ECFP + RBS + terminator + LVA tag	E. coli TOP10	pSB1AK3	1/11G
[http://partsregistry.org/Part:BBa_E0432 Bba_E0432]	EYFP + RBS + terminator + LVA tag	E. coli TOP10	pSB1A2	4/11C

To ensure successful cloning of the PCR products (*a→g*), (*a→d*) and (*d→g*) from 10/07, 1ul of GoTaq was added to each 50ul PCR reaction and extended for 10 min at 72°C. Four large lanes of (*a→g*) were run and each had a band of interest of ~8kb . One lane of each (*a→d*) and (*d→g*) was ran and only that of (*a→d*) had a band of interest of ~3kb. Each gel was 1% agarose, 50v for 40 min).
(*a→g*) and (*a→d*) were gel extracted (see Protocol 11 ), cloned into TOPO vectors(see Protocol 12 ), transformed (see Protocol 13 )and grown overnight at 37°C, each reaction divided into 100ul and ~200ul per plate.

Each plate of transformations of (*a→g*) and (*a→d*) grew several hundred colonies. Eight colonies from plates spread with 100ul of reaction were selected for colony PCR using Reddymix and Touch 2 program (see Protocol 9). Each selected colony was used in a reaction with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1. No expected bands were of 2kb were observed in either reaction.
Overnights of Colonies 1 and 2 from each of the above plates (spread with 100ul of transformants) were inoculated overnight in carb LB.

Miniprepped overnights according to QIAGEN manual (see Protocol 5), and were labelled as follows:

PCR was done for each of the above minipreps with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1, used Reddymix and Touch2 (see Protocol 9). There is no evidence of a 7 gene operon insert in any of the colonies.
Lynsey repeated amplification of XylR and XylR+Pr with KOD polymerase (see Protocol 9) using the primers listed below and subsequently cloned into TOPO vector (see Protocol 12) and transformed TOP10 cells (see Protocol 13).
- Primer Pairs:
  - XylR Prefix + XylR Suffix
  - Pr Prefix + XylR Suffix
  - Pr_for_2 + Xylr_rev_2

Maija performed PCR based site-directed mutagenesis for (*m*) to correct one PstI site and (*s*) to correct two PstI sites.

Biobrick + Construction Vector	Primer Pair
(m) + 3/20G	(m)_SDM_PstI_1 for + (m)_SDM_PstI rev
(m) + 4/6B	(m)_SDM_PstI_1 for + (m)_SDM_PstI rev
(s) + 3/20G	(s)_SDM_PstI_for + (S)_SDM_PstI_1 rev
(s) + 4/6B	(s)_SDM_PstI_for + (S)_SDM_PstI_1 rev
(s) + 3/20G	(s)_SDM_PstI_2 for + (s)_SDM_PstI_2_rev
(s) + 4/6B	(s)_SDM_PstI_2 for + (s)_SDM_PstI_2_rev

KOD polymerase was used during the mutagenesis PCR, (see Protocol 9) and (seeProtocol 17)

Repeated digests from 9/08 with PstI and EcoRI of minipreps thought to contain (*a→g*). There is a possibility that colony 4/1 contains the (*a→g*) insert.
5 ml overnight cultures done with LB kanamycin or LB carbenicillin from the transformed cell colonies 1-18 after the first round of the site-directed mutagenesis in order to get rid of PstI sites inside our becoming BioBrick genes (*m*) and (*s*).

Christine did more PCR of the minipreps of the colonies suspected of containing the 7 gene operon using Reddy mix, Touch 2 and the pimer combinations below for each miniprepped colony (see Protocol 9). The result gave no evidence of the insert being present.
- *M13_For and *B_EcoRI_SDM_RevI
- *M13_Rev and *B_EcoRI_SDM_RevI
Maija miniprepped the mutagenised cell cultures 1-18 and performed a PstI restriction digest with Roche's PstI restriction enzyme to verify whether the mutagenesis worked or not.
On the same 1% agarose gel Maija run the original plasmids which had not been mutagenised but had been digested with the same Roche's PstI restriction enzyme. The original plasmids were the following:
- (*m*) + 4/6B
- (*m*) + 3/20G
- (*s*) + 4/6B
- (*s*) + 3/20G

@@ Line 211: / Line 211: @@
 #*[https://2007.igem.org/Glasgow/Wetlab/Orders *M13_Rev] and [https://2007.igem.org/Glasgow/Wetlab/Orders *B_EcoRI_SDM_RevI]
 #Maija miniprepped the mutagenised cell cultures 1-18 and performed a PstI restriction digest with Roche's PstI restriction enzyme to verify whether the mutagenesis worked or not.
-#On the same 1% agarose gel Maija run original plasmids which had not been mutagenised but had been digested with the same Roche's PstI restriction enzyme. The original plasmids were the following:
+#On the same 1% agarose gel Maija run the original plasmids which had not been mutagenised but had been digested with the same Roche's PstI restriction enzyme. The original plasmids were the following:
 #*(*m*) + 4/6B
 #*(*m*) + 3/20G
 #*(*s*) + 4/6B
 #*(*s*) + 3/20G