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Formal Lab Session - 3rd September 2009

Chassis team

Today we continued the lab work on our PCR primers. We again attempted to use two pairs of our primers; the ones to amplify the sleB and cwlJ genes from the B. subtilis genome. We prepared the solutions needed, and followed the PCR protocol. We rehydrated our primers, and diluted them accordingly. The TMs were lowered, following yesterday's failed attempt to 50 and 45 degrees. We prepared four lots of solutions, two with genomic DNA and two without (our controls), containing 2 and 3ul of primer. They were loaded into the PCR machine, which ran for around 2 hours 23 minutes.

When we ran the products on a gel.

Test concentration of PCR primers

Using NanoDrop spectrophotometer

• sleBForward: 202.8ng/ul 19pmol/ul
• sleBReverse: 272.3ng/ul 30pmol/ul
• cwlJForward: 318.8ng/ul 28pmol/ul
• cwlJReverse: 248.9ng/ul 27pmol/ul

Redo PCR

Exp. PCR experiment for gene cwlJ and sleB

No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
1	24	15	sleBForward 3ul	sleBReverse 2ul	168 DNA 5ul	MolTaq (1u/ul) 1ul	50
2	29	15	sleBForward 3ul	sleBReverse 2ul	0	MolTaq (1u/ul) 1ul	50
3	24	15	sleBForward 3ul	sleBReverse 2ul	168 DNA 5ul	MolTaq (1ul)	50
4	29	15	sleBForward 3ul	sleBReverse 2ul	0	MolTaq (1u/ul) 1ul	50
5	23	15	cwlJForward 3ul	cwlJReverse 3ul	168 DNA 5ul	MolTaq (1u/ul) 1ul	50
6	28	15	cwlJForward 3ul	cwlJReverse 3ul	0	MolTaq (1u/ul) 1ul	50
7	23	15	cwlJForward 3ul	cwlJReverse 3ul	168 DNA 5ul	MolTaq ((1u/ul) 1ul	50
8	28	15	cwlJForward 3ul	cwlJReverse 3ul	0	MolTaq (1u/ul) 1ul	50

PCR procedure

• No.1,2,5,6:

95C 2min -->[95C 30S --> 50C 30S --> 75C 1.5min] 30 runs --> 75C 5min --> store at 4C

• No.3,4,7,8:

95C 2min -->[95C 30S --> 45C 30S --> 75C 1.5min] 30 runs --> 75C 5min --> store at 4C

PCR result

• Still no bands when we run the PCR result on 0.8% agarose gel.

conclusion

• Try to change the Tag enzyme instead of MotTag
• Try to redesign Primers

Stochastic switch team

Summary

Today we ran the PCR products for sac, ara and sspb on a 0.8% gel with wide wells, which we then cut out using the transilluminator. Goksel also did a ligation of the sspb brick that was purified from the gel using the sigma gel extraction protocol. We ligated the product with the pSB1AT3 plasmid backbone prepared last week. Both the sspb insert and the backbone were cut with EcoRI and SpeI. Today Arun also ran another PCR including our ara region, it worked well so we believe we can use the resulting PCR product for a digest and a ligation.

sac, araR and sspB PCR products were run on the gel. The order of the weels is: ladder, sac, araR, sspB, ladder.

Ligation

• Add water, 14ul
• Add ligation buffer, 3ul
• Mix well
• Add the sspB insert, 10ul
• Add the psB1AT3 plasmid backbone, 2ul
• Add the ligase, 1ul
• Vortex, pippet up&down
• Store at the fridge overnight suspending in water

We also did a control without the insert to measure the background level of religation of the vector. It should be zero.

Promoter Library Sub-Project

Introduction

From the previous lab session the products of the PCR reactions attempting to amplify the sigA promoter library (synthesized sequences of the sigA library containing degeneracies) were run through 1.5% agarose gel in electrophoresis. Analysis of the gel showed that although there were hazy bands forming, these may have not been the result of the PCR products and may have been caused by the primer-dimer complexes formed. The promoter fragments are roughly 100bp however, meaning that there may be little distinction between the promoters and the primer-dimers in 1.5% agarose gel! In light of this experiment, today's task will involve cleaning up the PCR products for another run of DNA gel electrophoresis tomorrow.

PCR Product Cleanup

The PCR Product Kit used to purify the PCR results was the GenElute™ PCR Clean-Up Kit, purchased from Sigma-Aldrich. The protocol used in this experiment is the one given with the kit. There were no changes to the protocol and the steps taken to perform the purification can be seen here:

• Took four Mini-prep Binding Columns and placed them into collection tubes; to each 0.5ml of Column Preparation Solution was added. The tubes were then centrifuged for 1 minute under 12,000g.

• Once the eluate in all of the collection tubes was removed, 200ul of Binding Solution was added to the four PCR Eppendorf tubes containing the PCR DNA (the four tubes containing 40ul of C0, V1, V2 and V3 PCR DNA). These four PCR DNA mixtures were then transferred to the four prepared MiniPrep Binding Columns and centrifuged for 1 minute under 13,000rpm. The eluate was discarded once again with the binding columns returned to their respective collection tubes.

• 0.5ml of Wash Solution was then added to the four binding columns. These binding columns (within the collection tubes) were then centrifuged for 1 minute under 13,000rpm with the resulting eluate discarded. The binding columns were then placed in their original collection tubes and centrifuged again at 13,000rpm; this time with no solutions added.

• The binding columns were then transferred to four new collection tubes (appropriately labelled) whilst the original collection tubes (plus eluate from the 2 minute centrifugation) were discarded. 50ul of Elution Solution was then added to the binding columns and allowed to sit within the tubes on the bench for 1 minute.

• The columns (within their new collection tubes) were then centrifuged for 1 minute under 13,000 rpm but this time it was the eluates which were kept and the binding columns discarded. The four collection tubes containing the eluated PCR products of C0, V1, V2 and V3 PCR reactions were then stored in the -20°C.

Conclusion

With the PCR products purified, another attempt at DNA gel electrophoresis under 1.5% agarose gel should confirm whether the PCR reactions to amplify our synthesized sigA promoter library has worked.

Metal Sensing Team

Introduction

Today we shall again run the products from yesterdays PCR reaction on agarose gel (through DNA gel electrophoresis) to determine whether we have been successful this time. If we are successful the team will purify the PCR product; if not the team will reattempt a PCR reaction.

Practical Outline

By the end of the day the Metal Sensing team hope to accomplish these objectives:

• Run fragments from PCR reaction (carried out yesterday) on gel through electrophoresis.
• If PCR reaction successful clean up products; if unsuccessful reattempt PCR reaction

Observations

We ran the products from yesterday's PCR reaction on gel and after about 30-40 minutes of DNA gel electrophoresis, the team analysed the gel using GelDoc:

The lanes of the gel were as follows:

• Lane 1 = blank
• Lane 2 = HindIII DNA ladder
• Lane 3 = czrA PCR product
• Lane 4 = czrA PCR control
• Lane 5 = ara PCR product
• Lane 6 = ara PCR control
• Lane 7 = Bacillus subtilis genomic DNA
• Lane 8 = Bacillus subtilis genomic DNA
• Lane 9 = Bacillus subtilis genomic DNA
• Lane 10 = HindIII DNA ladder

As far as the Metal Sensing team are concerned the PCR reaction involving the gene czrA did not work at all - this means we need to reattempt the PCR reaction. Meanwhile the Stochastic Switch team's ara coding sequence PCR did work.

Procedure

PCR

Today, after having run the gel for yesterdays PCR products, we are carrying out another PCR to amplify czrA gene which didn't work yet at different annealing temperatures: 40C 45C and 50C.

Exp.No.5 PCR experiment plan

No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
1	24	15	pMA1 2.5ul	pMA2 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
2	29	15	pMA1 2.5ul	pMA2 2.5ul	0	MolTaq (1ul)	50
3	24	15	pMA1 2.5ul	pMA2 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
4	29	15	pMA1 2.5ul	pMA2 2.5ul	0	MolTaq (1ul)	50
5	24	15	pMA1 2.5ul	pMA2 2.5ul	168 DNA 5ul	MolTaq (1ul)	50
6	29	15	pMA1 2.5ul	pMA2 2.5ul	0	MolTaq (1ul)	50

PCR conditions:

For sample 1 and 2

1- 95C for 2min
2- 95C for 30sec
3- 40C for 1min30sec 
4- 75C for 2min
    Step 2-3-4 x 30
5- 75C for 5min
6- 4C Pause

For sample 3 and 4

1- 95C for 2min
2- 95C for 30sec
3- 45C for 1min30sec 
4- 75C for 2min
    Step 2-3-4 x 30
5- 75C for 5min
6- 4C Pause

For sample 5 and 6

1- 95C for 2min
2- 95C for 30sec
3- 50C for 1min30sec  
4- 75C for 2min
    Step 2-3-4 x 30
5- 75C for 5min
6- 4C Pause

July M T W T F S S     [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_July_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_July_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_July_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_July_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_July_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/14_July_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_July_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_July_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_July_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_July_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_July_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_July_2009&action=edit 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_July_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_July_2009 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_July_2009&action=edit 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_July_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_July_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_July_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_July_2009 30] [http://2009.igem.org/Team:Newcastle/Labwork/31_July_2009 31]

August M T W T F S S           [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_August_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_August_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_August_2009&action=edit 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_August_2009 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_August_2009 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_August_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_August_2009 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_August_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_August_2009&action=edit 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_August_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_August_2009 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_August_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_August_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_August_2009 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_August_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_August_2009&action=edit 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_August_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_August_2009 18] [http://2009.igem.org/Team:Newcastle/Labwork/19_August_2009 19] [http://2009.igem.org/Team:Newcastle/Labwork/20_August_2009 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_August_2009 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_August_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_August_2009&action=edit 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_August_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_August_2009 25] [http://2009.igem.org/Team:Newcastle/Labwork/26_August_2009 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_August_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_August_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_August_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_August_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_August_2009&action=edit 31]

September M T W T F S S   [http://2009.igem.org/Team:Newcastle/Labwork/1_September_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_September_2009 2] [http://2009.igem.org/Team:Newcastle/Labwork/3_September_2009 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_September_2009 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_September_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_September_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_September_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_September_2009 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_September_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_September_2009 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_September_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_September_2009&action=edit 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_September_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_September_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_September_2009 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_September_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_September_2009 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_September_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_September_2009&action=edit 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_September_2009 21] [http://2009.igem.org/Team:Newcastle/Labwork/22_September_2009 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_September_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_September_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_September_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_September_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_September_2009&action=edit 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_September_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_September_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_September_2009 30]

October M T W T F S S       [http://2009.igem.org/Team:Newcastle/Labwork/1_October_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_October_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_October_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_October_2009&action=edit 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_October_2009 5] [http://2009.igem.org/Team:Newcastle/Labwork/6_October_2009 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_October_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_October_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_October_2009 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_October_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_October_2009&action=edit 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_October_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_October_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_October_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_October_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_October_2009 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_October_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_October_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_October_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_October_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_October_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_October_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_October_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/24_October_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_October_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_October_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_October_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/28_October_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_October_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_October_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_October_2009&action=edit 31]